JP7004637B2 - Chiral control - Google Patents

Description

Mutual reference to related applications This application is written in US Patent Application No. 61 / 671,655 filed on July 13, 2012, and No. 61 / 671,656 filed on July 13, 2012. Claiming the priority of No. 61 / 671,722 filed on July 14, 2012 and No. 61 / 671,724 filed on July 14, 2012, the full text of each application is , Incorporated herein by reference.

Technical Field Oligonucleotides are useful in therapeutic, diagnostic, research and nanomaterial applications.
Background Technique The therapeutic use of natural nucleic acids (eg, unmodified DNA or RNA) can be limited, for example, due to extracellular and intracellular nucleases and / or their low cellular permeability and low cellular distribution. In addition, in vitro studies have shown that binding affinity, specific binding sequences to complementary RNA (Cosstick and Eckstein, 1985; LaPlanche et al., 1986; Latimer et al., 1989; Hacia et al., 1994; Mesmaeker et. It was found that the properties of antisense oligonucleotides, such as al., 1995), and nuclease stability, could be influenced by the absolute stereochemical arrangement of the phosphorus atom (Cook et al., US Patent Publication No. 005599797). Therefore, new and improved oligonucleotide compositions are needed.

The present invention embraces the recognition that there is a need for chirally controlled oligonucleotide compositions and novel synthetic methods thereof. The present invention identifies the cause of certain problems in conventional methods of chiral oligonucleotide synthesis, including problems that interfere with the synthesis of fully chiral controlled compositions, in particular compositions containing multiple oligonucleotide types. Include.

In some embodiments, the invention provides a chirally controlled oligonucleotide composition.

In some embodiments, the invention provides a method for producing a chiral-controlled oligonucleotide and a chiral-controlled oligonucleotide composition.

In some embodiments, the invention provides a method of using a chiral-controlled oligonucleotide and a chiral-controlled oligonucleotide composition.

All publications and patents cited in this application are incorporated herein by reference in their entirety.

Definition Aliphatic: As used herein, the term "aliphatic" or "aliphatic group" is fully saturated or one or more unsaturated, having one binding point to another molecule. A straight chain (ie, non-branched) or branched chain containing units, a substituted or unsubstituted hydrocarbon chain, or a monocyclic hydrocarbon containing a fully saturated or one or more unsaturated units but not aromatic. It means a polycyclic hydrocarbon (also referred to herein as "carbon ring", "aliphatic" or "cycloalkyl"). In some embodiments, the aliphatic group comprises 1-50 aliphatic carbon atoms. Unless otherwise specified, an aliphatic group contains 1 to 10 aliphatic carbon atoms. In some embodiments, the aliphatic group comprises 1-6 aliphatic carbon atoms. In some embodiments, the aliphatic group comprises 1-5 aliphatic carbon atoms. In other embodiments, the aliphatic group comprises 1 to 4 aliphatic carbon atoms. In yet another embodiment, the aliphatic group comprises 1 to 3 aliphatic carbon atoms, and in yet another embodiment, the aliphatic group comprises 1 to 2 aliphatic carbon atoms. In some embodiments, the "alicyclic" (or "carbon ring" or "cycloalkyl") is a fully saturated or one or more unsaturated unit having one binding point to another molecule. Represents monocyclic or bicyclic C ₃ to C ₁₀ hydrocarbons that are contained but not aromatic. In some embodiments, the "alicyclic" (or "carbon ring" or "cycloalkyl") is a fully saturated or one or more unsaturated unit having one binding point to another molecule. Represents monocyclic C ₃ to C ₆ hydrocarbons that are contained but not aromatic. Suitable aliphatic groups include linear or branched chain, substituted or unsubstituted alkyl groups, alkenyl groups, alkynyl groups, and composites thereof such as (cycloalkyl) alkyl, (cycloalkenyl) alkyl or (cycloalkyl) alkenyl. The body is mentioned, but not limited to this.

Alkylene: The term "alkylene" refers to a divalent alkyl group. The "alkylene chain" is a polymethylene group, that is,-(CH ₂ ) _n- , in which n is a positive integer, preferably 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2. ~ 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are substituted with a substituent. Suitable substituents include those described below for substituted aliphatic groups.

Alkenylene: The word "alkenylene" stands for divalent alkenyl group. A substituted alkenylene group is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are substituted with a substituent. Suitable substituents include those described below for substituted aliphatic groups.

Animals: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" represents a human at any stage of development. In some embodiments, "animal" represents a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (eg, rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and / or helminths. In some embodiments, the animal can be a transgenic animal, a genetically modified animal, and / or a clone.

Approximately: As used herein, the term "approximately" or "about" as used to describe a number is generally used in general unless otherwise specified or otherwise explicit from the context (such number is less than 0% or 100). Including numbers within the range of 5%, 10%, 15%, or 20% in either direction (larger or smaller) of the number (unless it is a possible value greater than%). think. In some embodiments, the use of the word "about" when referring to a dose means ± 5 mg / kg / day.

Aryl: The term "aryl", used alone or as part of a large portion as "aralkyl", "aralkossi", or "aryloxyalkyl", has at least one ring of structure aromatic and is of the structure. Represents monocyclic and bicyclic ring structures having a total of 5-14 ring members, each ring containing 3-7 ring members. The word "aryl" may be used interchangeably with the word "aryl ring". In certain embodiments of the invention, "aryl" represents an aromatic ring structure comprising, but not limited to, phenyl, biphenyl, naphthyl, anthracyl, etc., which may have one or more substituents. .. Groups in which the aromatic ring is condensed with one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, are also referred to herein by the term "aryl". Is included in the range of.

Characteristic Parts: As used herein, the phrase "characteristic part" of a protein or polypeptide, as a whole, is a sequence of amino acids, or a sequence of amino acids that is characteristic of a protein or polypeptide. It contains a set. Each such continuous chain will generally contain at least two amino acids. Moreover, one of ordinary skill in the art will recognize that at least 5, 10, 15, 20 or more amino acids are usually required to be characteristic of the protein. In general, characteristic moieties share at least one functional feature with the relevant intact protein, in addition to the particular sequence homology described above.

Characteristic sequence: A "characteristic sequence" is a sequence found in all members of a family of polypeptides or nucleic acids and can therefore be used by one of ordinary skill in the art to define the members of that family.

Characteristic Structural Elements: The term "characteristic structural elements" refers to distinct structural elements found throughout the family of polypeptides, small molecules, or nucleic acids (eg, skeletal structures, sets of suspensions, etc.). It represents an array element, etc.) and can therefore be used by those of skill in the art to define the members of the family.

Equivalence: The term "equivalent" is used to describe two (or more) sets of states or environments that are sufficiently similar to each other so that the results obtained or the observed phenomena can be compared. As used herein. In some embodiments, an equivalent set of states or environments is characterized by a plurality of substantially the same features and one or a few modified features. Those skilled in the art will be characterized by a sufficient number and type of substantially the same features, and differences in the results or observed phenomena obtained under different sets of conditions or environments will change the set of conditions. You will recognize that they are equivalent to each other when you can justify the reasonable conclusion that the difference in the features is due to or indicates.

Dosage Plan: As used herein, a "dose plan" or "treatment regimen" is usually divided by a period of time and a set of unit doses (usually one) administered individually to a subject. Above). In some embodiments, a given therapeutic agent has a required dosing regimen that may include one or more doses. In some embodiments, the dosing regimen comprises multiple doses, each of which is separated from each other over a period of the same length; in some embodiments, the dosing regimen separates multiple doses and separate doses. Includes at least two different periods. In some embodiments, the total dose within the dosing regimen is the same unit dose. In some embodiments, the different doses within the dosing regimen are different amounts. In some embodiments, the dosing regimen comprises a first dose at the first dose and then one or more additional doses at a second dose that is different from the first dose. In some embodiments, the dosing regimen comprises a first dose at the first dose and then one or more additional doses at the same second dose as the first dose.

Equivalent Agents: Reading this disclosure, one of ordinary skill in the art will recognize that the scope of useful agents in the context of the present invention is not limited to those specifically mentioned or exemplified herein. Specifically, one of ordinary skill in the art recognizes that an activator usually has a structure consisting of a skeleton and an attached suspension, so a simple modification of such skeleton and / or suspension is the activity of the agent. You will understand that it does not significantly change. For example, in some embodiments, the substitution of one or more suspensions having groups of equivalent three-dimensional structure and / or chemical reaction properties is the substitution of a substituted compound or moiety equivalent to the parent reference compound or moiety. Can be generated. In some embodiments, the addition or separation of one or more suspensions may produce a substituted compound equivalent to the parent reference compound. In some embodiments, alteration of the skeletal structure by addition or separation of, for example, a small number of bonds (usually 5, 4, 3, 2 or less, or one bond, and often only a single bond) is a parental reference compound. Can produce compounds that are equivalently substituted with. In many embodiments, the equivalent compound is, for example, using readily available substances, reagents and conventional or provided synthetic procedures, by the method shown in the general reaction scheme below, or by modifications thereof. Can be synthesized. In these reactions, changes that are known in their own right but not mentioned here are also available.

Equivalent Dosage: The term "equivalent dose" is used herein to compare doses of different pharmaceutically active agents that produce the same biological results. Dosages of two different agents are considered "equivalent" to each other, according to the present invention, if they achieve comparable levels or degrees of biological results. In some embodiments, equivalent doses of different pharmaceuticals used in accordance with the present invention are determined using the in vitro and / or in vivo assays described herein. In some embodiments, one or more lysosome activators used in accordance with the present invention are utilized at a dose equivalent to the dose of the reference lysosome activator; in some embodiments, the reference lysosome activator of such interest. Are small molecule allosteric activators (eg, pyrazolpyrimidines), imminosugars (eg, isofagomin), antioxidants (eg, n-acetylcysteine), and regulators of cell transport (eg, n-acetylcysteine). For example, it is selected from the group consisting of Rab1a polypeptide).

Heteroaliphatic: The term "heteroaliphatic" refers to an aliphatic group in which one or more units selected from C, CH, CH ₂ or CH ₃ are independently substituted with heteroatoms. In some embodiments, the heteroaliphatic group is heteroalkyl. In some embodiments, the heteroaliphatic group is a heteroalkenyl.

Heteroaryl: The terms "heteroaryl" and "heteroar-" used alone or as part of a large portion, eg, "heteroaralkyl", or "heteroararcosis", are 5-10 ring atoms, Preferably, it has 5, 6, or 9 ring atoms; it has 6, 10, or 14 π electrons shared in the cyclic sequence; and in addition to the carbon atom, 1 Represents a group having up to 5 heteroatoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur and includes any oxidized form of nitrogen or sulfur, as well as a quaternized form of any basic nitrogen. Heteroaryl groups include thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isooxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridadinyl, pyrimidinyl, pyrazinyl, indridinyl, prynyl, naphthyldinyl, and However, it is not limited to this. As used herein, the terms "heteroaryl" and "heteroar-" mean that a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings with radicals or bonding points on the aromatic ring. Including radicals. Examples include, but are not limited to, indolyl, isoindrill, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzoimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolidinyl, carbazolyl, acridinyl, phenazinyl, phenazinyl. Examples include phenoxadinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido [2,3-b] -1,4-oxadin-3 (4H) -one. The heteroaryl group can be monocyclic or bicyclic. The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring", "heteroaryl group", or "aromatic heterocycle", any of which may be substituted. including. The term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl, the alkyl and the heteroaryl moiety may be independently substituted.

Heteroatom: The term "heteroatom" refers to one or more oxygen, sulfur, nitrogen, phosphorus, or silicon (oxidized forms of either nitrogen, sulfur, phosphorus, or silicon; quaternized forms of any basic nitrogen. Or; means a heterocycle, eg, N (in 3,4-dihydro-2H-pyrrolidyl), NH (in pyrrolidinyl) or NR ⁺ (including substitutable nitrogen in N-substituted pyrrolidinyl). ..

Heterocycles: As used herein, the terms "heterocycle,""heterocyclyl,""heterocyclicradical," and "heterocyclic ring" are used interchangeably. Stable 3- to 7-membered monocyclic or 7 to 10-membered, saturated or partially unsaturated and having one or more, preferably 1 to 4 of the above heteroatoms in addition to carbon atoms. Represents a bicyclic heterocyclic part. When used to describe a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. For example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen is such as in N (3,4-dihydro-2H-pyrrolill). ), NH (as in pyrrolidinyl) or ⁺ NR (as in N-substituted pyrrolidinyl).

The heterocycle can be attached to its side group at any heteroatom or carbon atom that provides a stable structure, and any ring atom may be substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl. , Dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinucridinyl. The terms "heterocycle", "heterocyclyl", "heterocyclyl ring", "heterocyclic group", "heterocyclic moiety", and "heterocyclic radical" are used interchangeably herein. The heterocyclyl ring comprises one or more aryl groups, heteroaryl groups, or groups that condense to an aliphatic ring, such as indolinyl, 3H-indrill, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, the radical or bond thereof. The points are on the heterocyclyl ring. The heterocyclyl ring can be monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted with heterocyclyl, the alkyl and heterocyclyl moieties may be substituted independently.

Intraperitoneal: As used herein, the terms "intraperitoneal administration" and "intraperitoneal administration" are understood in its art to describe administration of a compound or composition into the peritoneum of a subject. Has a meaning to be.

In vitro: As used herein, the term "in vitro" is used not in an organism (eg, an animal, a plant, and / or a microorganism) but in an artificial environment, such as a test tube or reactor. , Represents an event that occurs in a cell culture medium or elsewhere.

In vivo: As used herein, the term "in vivo" refers to an event that occurs in an organism (eg, an animal, a plant, and / or a microorganism).

Lower Alkyl: The term "lower alkyl" refers _to a C1-4 straight chain or branched chain alkyl group. By way of example, the lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

Lower haloalkyl: The term "lower haloalkyl" refers _to a C1-4 straight chain or branched chain alkyl group substituted with one or more halogen atoms.

May Be Substituted: As described herein, the compounds of the invention may include "may be substituted" moieties. In general, the word "substituted" means that one or more hydrogens in the designated moiety are substituted with the appropriate substituents, with or without the word "may". Unless otherwise indicated, a "optionally substituted" group is at each substitutable position of any given structure when one or more positions in any given structure can be the same or different from position to position. It may have a suitable substituent. The combination of substituents considered according to the present invention preferably results in the production of stable or chemically possible compounds. As used herein, the term "stable" refers to their manufacture, detection, and, in certain embodiments, their recovery, purification, and use for one or more purposes disclosed herein. Represents a compound that does not change substantially when in a state that enables.

Suitable monovalent substituents _on the substitutable carbon atoms of the "may be substituted" groups are independently halogens;-(CH ₂ ) 0-4 R ^○ ;-(CH ₂ ) 0-4 _. OR ^○ ; -O (CH ₂ ) _{0 to 4} R ^○ , -O- (CH ₂ ) _{0 to 4} C (O) OR ^○ ;-(CH ₂ ) _{0 to 4} CH (OR ^○ ) ₂ ;-(CH) ₂ ) _{0 to 4} SR ^○ ; may be replaced with R ^○ (CH ₂ ) _{0 to 4} Ph; may be replaced with R ^○ (CH ₂ ) _{0 to 4} O (CH ₂ ) _{0 to 1} Ph; may be substituted with R ^○ CH = CHPh; may be substituted with R ^○ (CH ₂ ) _{0 to 4} O (CH ₂ ) _{0 to 1} -pyridyl; -NO ₂ ; -CN;- N ₃ ; (CH ₂ ) _{0 to 4} N (R ^○ ) ₂ ;-(CH ₂ ) _{0 to 4} N (R ^○ ) C (O) R ^○ ; -N (R ^○ ) C (S) R ^○ ; -(CH ₂ ) _{0 to 4} N (R ^○ ) C (O) NR ^○ ₂ ; N (R ^○ ) C (S) NR ^○ ₂ ;-(CH ₂ ) _{0 to 4} N (R ^○ ) C (O) ) OR ^○ ; -N (R ^○ ) N (R ^○ ) C (O) R ^○ ; N (R ^○ ) N (R ^○ ) C (O) NR ^○ ₂ ; N (R ^○ ) N (R ^○ ) C (O) OR ^○ ;-(CH ₂ ) _{0 to 4} C (O) R ^○ ; -C (S) R ^○ ;-(CH ₂ ) _{0 to 4} C (O) OR ^○ ;-(CH ₂ ) _{0 to 4} C (O) SR ^○ ; (CH ₂ ) _{0 to 4} C (O) OSiR ^○ ₃ ;-(CH ₂ ) _{0 to 4} OC (O) R ^○ ; -OC (O) (CH ₂ ) _{0 ~ 4 SR-} , SC (S) SR ^○ ;-(CH ₂ ) 0-4 SC ( _O ) R ^○ ;-(CH ₂ ) 0-4 C (O) NR ^○ ₂ ; -C (S) NR ^○ ₂ ; -C (S) SR ^○ ; -SC (S) SR ^○ , (CH ₂ ) 0-4 OC ( _O ) NR ^○ ₂ ; C (O) N (OR ^○ ) R ^○ ; -C (O) C (O) R ○; -C (O) CH2C (O) R ○; -C (NOR ○) R ^○ ; (CH ₂ ) 0-4 SSR ^○ ;-(CH ₂ ) 0-4 S _{( O} ) ₂ R ^○ ;-(CH ₂ ) 0-4 S ( _O ) ₂ OR ^○ ;-(CH ₂ ) 0-4 OS ( _O ) ₂ R ^○ ;-S (O) ₂ NR ^○ ₂ ; (CH ₂ ) _0-4 S (O) R ^○ ; N (R ^○ ) S (O) ₂ NR ^○ ₂ ; -N (R ^○ ) S (O) ₂ R ^○ ; -N (OR ^○ ) R ^○ ; -C (NH) NR ^○ ₂ ; -P (O) ₂ R ^○ ; P (O) R ^○ ₂ ; OP (O) R ^○ ₂ ; -OP (O) (OR ^○ ) ₂ ; -SiR ○ ₃ ;-(C _{1 to 4} ) Linear or branched alkylene) ON (R ^○ ) ₂ ; or-(C _1-4 linear or branched alkylene) C (O) ON (R ^○ ) ₂ (In the formula, each R ^○ is , May be substituted as follows, independently, hydrogen, C _1-6 aliphatic, -CH ₂ Ph, -O (CH ₂ ) _0-1 Ph, -CH _2- (5-6 member hetero). Aryl ring) or an aryl ring with 0-4 heteroatoms selected independently of nitrogen, oxygen, or sulfur, or a 5- to 6-membered saturated, partially unsaturated, or despite the above definition. , Two R ^○ appearing independently may be substituted as follows together with their intervening atoms, 0-4 independently selected from nitrogen, oxygen, or sulfur. It is 3- to 12-membered saturated with heteroatoms, partially unsaturated, or forms an aryl monocyclic or bicyclic ring).

Appropriate monovalent substituents on R ^○ (or rings formed by two independently appearing R ^○ together with their intervening atoms) are independently halogen,-(CH ₂ ). _{0 to 2} R ^● ,-(Halo R ^● ),-(CH ₂ ) _{0 to 2} OH,-(CH ₂ ) _{0 to 2} OR ^● ,-(CH ₂ ) _{0 to 2} CH (OR ^● ) ₂ ; O (Halo R ^● ), -CN, -N ₃ ,-(CH ₂ ) _{0 to 2} C (O) R ^● ,-(CH ₂ ) _{0 to 2} C (O) OH,-(CH ₂ ) _{0 to 2} C (O) OR ^● ,-(CH ₂ ) _{0 to 2} SR ^● ,-(CH ₂ ) _{0 to 2} SH,-(CH ₂ ) _{0 to 2} NH ₂ ,-(CH ₂ ) _{0 to 2} NHR ^● , -(CH ₂ ) _{0 to 2} NR ^● ₂ , -NO ₂ , -SiR ^● ₃ , -OSiR ^● ₃ , C (O) SR ^● ,-(C _1-4 linear or branched alkylene) C (O) OR ^● or -SSR ^● (In the formula, each R ^● is unsubstituted or substituted with only one or more halogens if preceded by “halo”, and independently, C _1-4 fatty groups. , -CH ₂ Ph, -O (CH ₂ ) _{0 to 1} Ph, or 5 to 6 member saturated, partially unsaturated, or 0 to 4 heteros selected independently of nitrogen, oxygen, or sulfur. (Selected from aryl rings with atoms). Suitable divalent substituents on the saturated carbon atom of R ^○ include = O and = S.

Suitable divalent substituents on the saturated carbon atom of the "optionally substituted" group include: = O, = S, = NNR ^* ₂ , = NNHC (O) R ^* , = NNHC ( O) OR ^* , = NNHS (O) ₂ R ^* , = NR ^* , = NOR ^* , -O (C (R ^* ₂ )) _2-3 O-, or -S (C (R ^* ₂ )) _{2 ~ 3} S- (in the formula, each R ^* appearing independently is hydrogen, the following substitutable C _1-6 aliphatic, or unsubstituted 5-6 member saturated, partially unsaturated, or nitrogen, It is selected from an aryl ring having 0 to 4 heteroatoms selected independently of oxygen or sulfur). As a suitable divalent substituent attached to the adjacent substitutable carbon of the "may be substituted" group: -O (CR ^* ₂ ) _2-3 O- (each R appearing independently in the equation). ^* Is hydrogen, the following substitutable C _1-6 aliphatic, or unsubstituted 5-6 member saturated, partially unsaturated, or 0-4 independently selected from nitrogen, oxygen, or sulfur. (Selected from aryl rings with heteroatoms).

Suitable substituents on the aliphatic group of R * include halogen, -R ^● ,-(halo R ^● ), -OH, -OR ^● , -O (halo R ^● ), -CN, -C (O). ) OH, -C (O) OR ^● , -NH ₂ , -NHR ^● , -NR ^● ₂ , or -NO ₂ (In the formula, each R ^● is unsubstituted or preceded by "halo". Substituted with only one or more halogens and independently, C _1-4 aliphatic, -CH ₂ Ph, -O (CH ₂ ) _{0 to 1} Ph, or 5 to 6 member saturated, partially non-existent It is an aryl ring having 0 to 4 heteroatoms selected independently of saturation or nitrogen, oxygen, or sulfur).

Suitable substituents on substitutable nitrogen of "may be substituted" groups are -R ^† , -NR ^† ₂ , -C (O) R ^† , -C (O) OR ^† , -C. (O) C (O) R ^† , -C (O) CH ₂ C (O) R ^† , -S (O) ₂ R ^† , S (O) ₂ NR ^† ₂ , -C (S) NR ^† ₂ , -C (NH) NR ^† ₂ , _or ^-N (R ^† ) S (O) ₂ R ^† ; Group, unsubstituted-OPh, or unsubstituted 5- to 6-membered saturated, partially unsaturated, or an aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or. Despite the above definition, two independently appearing R ^† , together with their intervening atoms, are 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. , Which are unsubstituted 3- to 12-membered saturated, partially unsaturated, or form an aryl monocyclic or bicyclic ring).

Suitable substituents on the aliphatic group of R ^† are independently halogen, -R ^● ,-(halo R ^● ), -OH, -OR ^● , -O (halo R ^● ), -CN,- C (O) OH, -C (O) OR ^● , -NH ₂ , -NHR ^● , -NR ^● ₂ , or -NO ₂ (In the formula, each R ^● is unsubstituted or preceded by "halo". When attached, it is substituted with only one or more halogens, and independently, C _1-4 aliphatic, -CH ₂ Ph, -O (CH ₂ ) _0-1 Ph, or 5-6 member saturated, partial. It is an aryl ring having 0 to 4 heteroatoms, which is essentially unsaturated or selected independently of nitrogen, oxygen, or sulfur).

Oral: As used herein, the terms "orally administered" and "orally administered" refer to oral administration of a compound or composition and have the meaning understood in the art. ..

Parenteral: As used herein, the terms "parenteral administration" and "parenteral administration" refer to a method of administration by conventional injection that is not intestinal or topical. Intravenous, intramuscular, arterial, intrathecal, intracapsular, intraocular, intracardiac, intracutaneous, intraperitoneal, transtracheal, subcutaneous, subepithelial, intra-articular, capsule Intrathecal, submucosal, intraspinal, and intrathoracic injections and infusions include, but are not limited to.

Partially unsaturated: As used herein, the term "partially unsaturated" refers to a ring moiety containing at least one double or triple bond. The term "partially unsaturated" is intended to include rings with multiple moieties of unsaturatedity, but is not intended to include the aryl or heteroaryl moieties as defined herein.

As used in, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a given therapeutic effect when administered to the appropriate population. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, with the following uses: oral administration, eg, liquid medicine (aqueous or non-aqueous solution or suspension). ), Tablets, such as those for oral, sublingual, and systemic absorption, ointments, powders, granules, pastes applied to the tongue; for example, as sterile solutions or suspensions, or sustained release formulations. For example, parenteral administration by subcutaneous, intramuscular, intravenous or epidural injection; topical administration, for example, as a cream, ointment, or sustained release patch or spray applied to the skin, lungs, or oral cavity; , Pessaries, creams, or foam powders in the vagina or rectum; under the tongue; in the eyes; transdermally; or nasally, lungs, and other mucosal surfaces.

Pharmaceutically acceptable: When used herein, the phrase "pharmaceutically acceptable" is, within sound medical judgment, commensurate with a reasonable benefit / risk ratio and is excessively harmful. Represents a compound, substance, composition, and / or dosage form suitable for use in contact with human and animal tissues without sex, irritation, allergic response, or other problems or complications.

Pharmaceutically Acceptable Carriers: As used herein, the term "pharmaceutically acceptable carrier" is used from one organ, or part of the body, to another organ, or part of the body. Means a pharmaceutically acceptable material, composition or medium such as a liquid or solid filler, diluent, excipient, or solvent-encapsulated material involved in transport or transport. Each carrier must be "acceptable" in the sense that it is compatible with the other ingredients of the formulation and is not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers are: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate and Derivatives; Tragant; Maltese; Gelatin; Starch; Excipients such as cocoa butter and suppository wax; Oils such as peanut oil, cottonseed oil, red flower oil, sesame oil, olive oil, corn oil and soybean oil; Glycers such as propylene glycol Polymers such as glycerin, sorbitol, manitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; exothermic free water; isotonic salt Water; Ringer's solution; Ethyl alcohol; pH buffered solution; Esters, polycarbonates and / or polyacid anhydrides; and other harmless compatible substances used in pharmaceutical formulations.

Pharmaceutically Acceptable Salts: As used herein, the term "pharmaceutically acceptable salt" refers to salts of such compounds suitable for use in the pharmaceutical context, ie, healthy. Suitable salts for use in contact with human and lower animal tissues, within medical judgment, commensurate with a reasonable benefit / risk ratio, without excessive harm, irritation, allergic response, etc. show. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., J. Pharmaceutical Sciences, 66: 1-19 (1977), describe in detail pharmaceutically acceptable salts. In some embodiments, pharmaceutically acceptable salts include adipates, alginates, ascorbinates, asparaginates, benzenesulfonates, benzoates, bicarbonates, borates, butyric acids. Salt, camphorate, cerebral sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptate, glyceroline Acids, Gluconate, Hemisulfate, Heptaneate, Hexate, Hydroiodide, 2-Hydroxy-Etan Sulfonate, Lactobionate, Lactate, Laurilate, Lauryl Sulfate, Apple Acids, maleates, malonic acids, methanesulphonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectinates, persulfates Salt, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, Examples include, but are not limited to, undecanoate, valerate, and the like. Typical alkaline or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. In some embodiments, the pharmaceutically acceptable salt has, where appropriate, halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, 1-6 carbon atoms. Examples include harmless ammonium, quaternary ammonium, and amine cations produced using counterions such as alkyl sulfonates and aryl sulfonates.

Prodrugs: Generally, "prodrugs" as used herein, as understood in the art, are metabolized throughout the body and have interesting activity (eg, eg) when administered to an organism. , Therapeutic or diagnostic) is the entity that delivers the agent. Typically, such metabolism causes the removal of at least one "prodrug moiety" so that the activator is produced. Various forms of "prodrugs" are well known in the art. An example of such a prodrug portion is:
a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, 42: 309-396, edited by K. Widder, et al. (Academic Press, 1985);
b) Prodrugs and Targeted Delivery, edited by J. Rautio (Wiley, 2011);
c) Prodrugs and Targeted Delivery, edited by J. Rautio (Wiley, 2011);
d) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen;
e) Bundgaard, Chapter 5 “Design and Application of Prodrugs”, by H. Bundgaard, p. 113-191 (1991);
f) Bundgaard, Advanced Drug Delivery Reviews, 8: 1-38 (1992);
g) Bundgaard, et al., Journal of Pharmaceutical Sciences, 77: 285 (1988); and h) Kakeya, et al., Chem. Pharm. Bull., 32: 692 (1984)
reference.

Like the other compounds described herein, the prodrug may be provided in any of various forms, such as crystalline form, salt form, etc. In some embodiments, the prodrug is provided as its pharmaceutically acceptable salt.

Protecting groups: As used herein, the term "protecting group" is well known in the art and is well known in the art, Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd edition, John Wiley & Sons, 1999. (This full text is incorporated herein by reference), including those described in detail. Nucleotides and nucleotides described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al. 06/2012 (the full text of Chapter 2 is incorporated herein by reference). Protecting groups specifically applicable to nucleotide chemistry are also included. Suitable amino protective groups include methylcarbamate, ethylcarbamate, 9-fluorenylmethylcarbamate (Fmoc), 9- (2-sulfo) fluorenylmethylcarbamate, 9- (2,7-dibromo) fluorenyl. Methylcarbamate, 2,7-di-t-butyl- [9- (10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)] Methylcarbamate (DBD-Tmoc), 4-methoxyphenancil Carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1- (1-adamantyl) -1-methylethyl carbamate ( Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl Carbamate (TCBOC), 1-methyl-1- (4-biphenylyl) ethyl carbamate (Bpoc), 1- (3,5-di-t-butylphenyl) -1-methylethyl carbamate (t-Bumeoc), 2- (2'-and 4'-pyridyl) ethyl carbamate (Pyoc), 2- (N, N-dicyclohexylcarboxamide) ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc) , Allylcarbamate (Allloc), 1-isopropylallylcarbamate (Ipaoc), cinnamylcarbamate (Coc), 4-nitrocinnamylcarbamate (Noc), 8-quinolylcarbamate, N-hydroxypiperidinylcarbamate, alkyldithiocarbamate , Benzylcarbamate (Cbz), p-methoxybenzylcarbamate (Moz), p-nitrobenzylcarbamate, p-bromobenzylcarbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzylcarbamate, 4-methylsulfinylbenzylcarbamate (Msz) ), 9-Anthylmethylcarbamate, Diphenylmethylcarbamate, 2-Methylthioethylcarbamate, 2-Methylsulfonylethylcarbamate, 2- (p-toluenesulfonyl) ethylcarbamate, [2- (1,3-dithianyl)] methylcarbamate (Dmoc), 4-Methylthiophenylcarbamate (Mtpc), 2,4-dimethylthiophenylcarbamate (Bmpc), 2-phosphonioethylcarbamate (Peoc), 2-triphenylphosphonioisopropylcarbamate (Ppoc), 1 , 1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acryloxybenzyl carbamate, p- (dihydroxyboryl) benzyl carbamate, 5-benzisooxazolylmethyl carbamate- , 2- (Trifluoromethyl) -6-chromonylmethylcarbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzylcarbamate, o-nitrobenzylcarbamate- , 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl) methyl carbamate, phenothiadinyl- (10) -carbonyl derivative, N'-p-toluenesulfonylaminocarbonyl derivative, N '-Phenylaminothiocarbonyl derivative, t-amylcarbamate, S-benzylthiocarbamate, p-cyanobenzylcarbamate, cyclobutylcarbamate, cyclohexylcarbamate, cyclopentylcarbamate , Cyclopropylmethylcarbamate, p-decyloxybenzylcarbamate, 2,2-dimethoxycarbonylvinylcarbamate, o- (N, N-dimethylcarboxamide) benzylcarbamate, 1,1-dimethyl-3- (N, N) -Dimethylcarboxamide) propylcarbamate, 1,1-dimethylpropynylcarbamate, di (2-pyridyl) methylcarbamate, 2-furanylmethylcarbamate, 2-iodoethylcarbamate, isobornylcarbamate, isobutylcarbamate, isonicotynylcarbamate, p- (p'-methoxyphenylazo) benzyl carbamate, 1-methylcyclobutylcarbamate, 1-methylcyclohexylcarbamate, 1-methyl-1-cyclopropylmethylcarbamate, 1-methyl-1- (3,5-dimethoxyphenyl) ) Ethylcarbamate, 1-methyl-1- (p-phenylazophenyl) ethylcarbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1- (4-pyridyl) ethylcarbamate, phenylcarbamate, p-( Phenylazo) benzyl carbamate, 2,4,6-tri-t-butylphenylcarbamate, 4- (tri) Methylammonium) benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropaneamide, picolinamide, 3-pyridylcarboxamide, N-benzoyl Phenylalanyl derivatives, benzamides, p-phenylbenzamides, o-nitrophenylacetamides, o-nitrophenoxyacetamides, acetacetamides, (N'-dithiobenzyloxycarbonylamino) acetamides, 3- (p-hydroxyphenyl) propanamides, 3- (o-Nitrophenyl) Propanamide, 2-Methyl-2- (o-Nitrophenoxy) Propanamide, 2-Methyl-2- (o-phenylazophenoxy) Propanamide, 4-Chlorobutaneamide, 3- Methyl-3-nitrobutaneamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, o- (benzoyloxymethyl) benzamide, 4,5-diphenyl-3-oxazoline-2-one, N-phthalimide, N-dithiasuccinateimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexane-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexane-2-one, 1-substituted 3 , 5-Dinitro-4-pyridone, N-methylamine, N-allylamine, N- [2- (trimethylsilyl) ethoxy] methylamine (SEM), N-3-acetoxypropylamine, N- (1-isopropyl-4) -Nitro-2-oxo-3-pyroolin-3-yl) amine, quaternary ammonium salts, N-benzylamine, N-di (4-methoxyphenyl) methylamine, N-5-dibenzosverylamine, N- Triphenylmethylamine (Tr), N-[(4-methoxyphenyl) diphenylmethyl] amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenyl Methyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picorylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylidene Amine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl) mesityl] methyleneamine, N- (N', N'-dimethylaminomethylene) amine, N, N'-isopropi Redenamine, Np-nitrobenzideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N- (5-chloro-2-hydroxyphenyl) phenylmethyleneamine, N-cyclohexylideneamine , N- (5,5-dimethyl-3-oxo-1-cyclohexenyl) amine, N-boran derivative, N-diphenylboric acid derivative, N- [phenyl (pentacarbonylchrome-or tungsten) carbonyl] amine, N -Copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkylphosphoroamidart Kind, dibenzylphosphoroamidet, diphenylphosphoroamidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro- 4-methoxybenzene sulfenamide, triphenylmethyl sulfenamide, 3-nitropyridine sulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4- Methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl- 4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-Pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4- (4' , 8'-Dimethoxynaphthylmethyl) benzenesulfonamide (DNMBS), benzylsulfoneamide, trifluoromethylsulfonamide, and phena Syl sulfonamides can be mentioned.

Suitable protected carboxylic acids include, but are not limited to, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids. Examples of suitable silyl groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl and the like. Examples of suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, tetrahydropyran-2-yl. Examples of suitable alkenyl groups include allyl. Examples of suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl. Examples of suitable arylalkyl groups are optionally substituted benzyls (eg, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2 , 6-Dichlorobenzyl, p-cyanobenzyl), and 2- and 4-picoryl.

Suitable hydroxyl protective groups include methyl, methoxymethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl) methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyl. Oxymethyl (PMBM), (4-Methoxyphenoxy) Methyl (p-AOM), Guayacol Methyl (GUM), t-Butoxymethyl, 4-Pentenyloxymethyl (POM), Syroxymethyl, 2-Methoxyethoxymethyl (MEM) , 2,2,2-Trichloroethoxymethyl, bis (2-chloroethoxy) methyl, 2- (trimethylsilyl) ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-Methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S, S-dioxide, 1-[(2-chloro-4-methyl) phenyl] -4-Methoxypiperazine-4-yl (CTMP), 1,4-dioxane-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a, 4,5,6,7,7a-octahydro-7 , 8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1- (2-chloroethoxy) ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxy Ethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2- (phenylselenyl) ethyl, t-butyl, allyl, p-chlorophenyl, p -Methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl , P-phenylbenzyl, 2-picoryl, 4-picoryl, 3-methyl-2-picoryl N-oxide, diphenylmethyl, p, p'-dinitrobenzhydryl, 5-dibenzosveryl, triphenylmethyl, α- Naftyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di (p-methoxyphenyl) phenylmethyl, tri (p-methoxyphenyl) methyl, 4- (4'-bromophenacyloxyphenyl) dipheni Lumethyl, 4,4', 4''-Tris (4,5-dichlorophthalimidephenyl) Methyl, 4,4', 4''-Tris (Rebrinoyloxyphenyl) Methyl, 4,4', 4'' -Tris (benzoyloxyphenyl) methyl, 3- (imidazole-1-yl) bis (4', 4''-dimethoxyphenyl) methyl, 1,1-bis (4-methoxyphenyl) -1'-pyrenylmethyl, 9 -Anthryl, 9- (9-phenyl) xanthenyl, 9- (9-phenyl-10-oxo) anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S, S-dioxide, trimethylsilyl (TMS) , Triethylsilyl (TES), Triisopropylsilyl (TIPS), Dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), Dimethyltexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS). , Tribenzylsilyl, Tri-p-xylsilylsilyl, Triphenylsilyl, Diphenylmethylsilyl (DPMS), t-Butylmethoxyphenylsilyl (TBMPS), Formic acid ester, Benzoylgic acid ester, Acetate ester, Chloroacetate ester, Dichloroacetic acid ester, Trichloroacetic acid ester, trifluoroacetic acid ester, methoxyacetic acid ester, triphenylmethoxyacetic acid ester, phenoxyacetic acid ester, p-chlorophenoxyacetic acid ester, 3-phenylpropionic acid ester, 4-oxopentanoic acid ester (levulinic acid ester), 4 , 4- (ethylene dithio) pentanoic acid ester (levrinoyl dithioacetal), pivalic acid ester, adamantate, crotonic acid ester, 4-methoxycrotonic acid ester, benzoic acid ester, p-phenyl benzoic acid ester, 2,4 , 6-trimethylbenzoic acid ester (mesitoate), alkylmethyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkylethyl carbonate, alkyl 2,2,2-trichloroethyl (Troc), 2- (Trimethylsilyl) ethyl (TMSEC), 2- (phenylsulfonyl) ethyl (Psec), 2- (triphenylphosphonio) ethyl (Peoc), alkylisobutyl carbonate, alkylvinyl carbonate, alkylallyl carbonate, alkylcarbonate p- Nitrophenyl, alkyl benzyl carbonate, alkyl carbonate p -Methoxybenzyl, alkyl 3,4-dimethoxybenzyl, alkyl o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, alkylthiocarbonate S-benzyl, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iode Sasal fragrance ester, 4-azidobutyric acid ester, 4-nitro-4-methylpentanoic acid ester, o- (dibromomethyl) benzoic acid ester, 2-formylbenzenesulfonic acid ester, 2- (methylthiomethoxy) ethyl, 4-( Methylthiomethoxy) butyric acid ester, 2- (methylthiomethoxymethyl) benzoic acid ester, 2,6-dichloro-4-methylphenoxyacetic acid ester, 2,6-dichloro-4- (1,1,3,3-tetramethylbutyl) ) Phenoxyacetic acid ester, 2,4-bis (1,1-dimethylpropyl) phenoxyacetic acid ester, chlorodiphenylacetic acid ester, isobutyric acid ester, monosuccinic acid ester, (E) -2-methyl-2-butenoic acid ester, o -(Methoxycarbonyl) benzoic acid ester, α-naphthoic acid, nitrate ester, alkyl N, N, N', N'-tetramethylphosphologiamidate, alkyl N-phenylcarbamate, borate ester, dimethylphosphinothionyl, Included are alkyl 2,4-dinitrophenyl sulfenates, sulfate esters, methanesulfonic acid esters (mesylic acid esters), benzylsulfonic acid esters, and tosylate (Ts). In order to protect 1,2- or 1,3-diols, the protective groups include methylene acetal, etylidene acetal, 1-t-butyl etylideneketal, 1-phenylethylideneketal, (4-methoxyphenyl). Echilidene acetal, 2,2,2-trichloroethiriden acetal, acetonide, cyclopentylideneketal, cyclohexylideneketal, cycloheptilideneketal, benziliden acetal, p-methoxybenzidylene acetal, 2,4-dimethoxybenzideneketal, 3, 4-Dimethoxybenzidene acetal, 2-nitrobenzidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene orthoester, 1-methoxyethylidene orthoester, 1-ethoxyethylidene orthoester, 1,2-dimethoxyethylidene orthoester, α- Methoxybenziliden orthoester, 1- (N, N-dimethylamino) etylidene derivative, α- (N, N'-dimethylamino) benziliden derivative, 2-oxacyclopentylidene orthoester, di-t-butylsilylene group (DTBS) ), 1,3- (1,1,3,3-Tetraisopropyldisiloxanilidene) derivative (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cyclic carbonates, Cyclic boronic acid esters, ethyl boronate, and phenyl boronate can be mentioned.

In some embodiments, the hydroxyl protecting group is acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1- (2-chloroethoxy) ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl (trityl), 4,4'-dimethoxytrityl, Trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylgiate ester, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate , Tocillate, Triflate, Trityl, Monomethoxytrityl (MMTr), 4,4'-Dimethoxytrityl, (DMTr) and 4,4', 4''-Trimethoxytrityl (TMTr), 2-Cyanoethyl (CE or Cne) ), 2- (trimethylsilyl) ethyl (TSE), 2- (2-nitrophenyl) ethyl, 2- (4-cyanophenyl) ethyl 2- (4-nitrophenyl) ethyl (NPE), 2- (4-nitro) Phenylsulfonyl) Ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2- (2-nitrophenyl) ethyl, butylthiocarbonyl , 4,4', 4''-tris (benzoyloxy) trityl, diphenylcarbamoyl, levulinyl, 2- (dibromomethyl) benzoyl (Dbmb), 2- (isopropylthiomethoxymethyl) benzoyl (Ptmt), 9-phenylxanthene -9-Il (pixyl) or 9- (p-methoxyphenyl) xanthin-9-yl (MOX). In some embodiments, each of the hydroxyl protecting groups is independently selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl and 4,4'-dimethoxytrityl. In some embodiments, the hydroxyl protecting group is selected from the group consisting of trityl, monomethoxytrityl and 4,4'-dimethoxytrityl groups.

In some embodiments, the phosphite protecting group is a group attached to an internucleotide phosphite bond throughout oligonucleotide synthesis. In some embodiments, the phosphite protecting group binds to the sulfur atom of an internucleotide phosphorothioate bond. In some embodiments, the phosphite protecting group binds to the oxygen atom of an internucleotide phosphorothioate bond. In some embodiments, the phosphite protecting group binds to the oxygen atom of an internucleotide phosphate bond. In some embodiments, the phosphite protecting group is 2-cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, o-nitrobenzyl, 2-(. p-Nitrophenyl) ethyl (NPE or Npe), 2-phenylethyl, 3- (N-tert-butylcarboxamide) -1-propyl, 4-oxopentyl, 4-methylthio-l-butyl, 2-cyano-1 , 1-dimethylethyl, 4-N-methylaminobutyl, 3- (2-pyridyl) -1-propyl, 2- [N-methyl-N- (2-pyridyl)] aminoethyl, 2- (N-formyl) , N-methyl) aminoethyl, 4- [N-methyl-N- (2,2,2-trifluoroacetyl) amino] butyl.

Protein: As used herein, the term "protein" refers to a polypeptide (ie, a chain of at least two amino acids attached to another by a peptide bond). In some embodiments, the protein contains only naturally occurring amino acids. In some embodiments, the protein comprises one or more non-naturally occurring amino acids (eg, moieties that form one or more peptide bonds with adjacent amino acids). In some embodiments, one or more residues of the protein chain comprises a non-amino acid moiety (eg, glycan, etc.). In some embodiments, the protein comprises, for example, more than one polypeptide chain bound by one or more disulfide bonds or associated by other means. In some embodiments, the protein comprises L-amino acids, D-amino acids, or both; in some embodiments, the protein comprises one or more amino acid modifications or analogs well known in the art. include. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. The term "peptide" is commonly used to describe a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than about 20 amino acids, or less than about 10 amino acids. To. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

Sample: As used herein, the term "sample" refers to a biological sample obtained or derived from a source of interest as described herein. In some embodiments, the source of interest comprises an organism such as an animal or human. In some embodiments, the biological sample comprises biological tissue or fluid. In some embodiments, the biological sample is bone marrow; blood; blood cells; ascites; tissue or needle biopsy sample; cell-containing body fluids; suspended nucleic acids; sputum; saliva: urine; cerebrospinal fluid, ascites; chest water; feces: Lymph fluid; Gynecological fluid; Skin swab; Vaginal swab; Oral swab; Nasal swab; Conduit lavage fluid or bronchial alveolar lavage fluid, etc. Other body fluids, secretions, and / or cells from it, others, or contain them. In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. For example, in some embodiments, the primary biological sample consists of a group consisting of biopsy (eg, needle aspiration or tissue biopsy), surgery, collection of body fluids (eg, blood, lymph, feces, etc.), and others. Obtained by the method of choice. In some embodiments, as is apparent from the context, the term "sample" refers to the primary sample by processing (eg, removal of one or more components thereof and / or addition of one or more agents to it). ) Represents the resulting preparation. For example, filtration using a semipermeable membrane. Such a "treated sample" may include, for example, a nucleic acid or protein obtained by extracting from the sample or treating the primary sample by techniques such as mRNA amplification or reverse transcription, isolation and / or purification of specific components, and the like.

Stereochemical isomers: As used herein, the term "stereochemical isomers" refers to different three-dimensional structures that are assembled with the same atoms bonded by the same series of bonds, but are not compatible. Represents a different compound that has. In some embodiments of the invention, the provided chemical composition may be, or may include, a pure synthesis of the individual steric chemical isomers of the compound; in some embodiments, it is provided. The chemical composition can be, or may contain, a mixture of two or more steric chemical isomers of the compound. In certain embodiments, such a mixture comprises the same amount of different stereochemical isomers; in certain embodiments, such mixture comprises different amounts of at least two different stereochemical isomers. In some embodiments, the chemical composition may comprise the entire diastereomeric and / or enantiomer of the compound. In some embodiments, the chemical composition may contain less than all diastereomers and / or enantiomers of the compound. In some embodiments, if a particular mirror isomer of the compound of the invention is desired, it can be synthesized, for example, by asymmetric synthesis or induction with a chiral auxiliary, resulting in diastereomers. The mar mixture separates and cleaves the auxiliary group to give the purely desired chiral isomer. Alternatively, if the molecule contains a basic functional group such as amino, the diastereomeric salt is produced with a suitable optically active acid and split, for example by fractional recrystallization.

Subject: As used herein, the term "subject" or "subject" means that the compound or composition provided is, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Represents any organism administered according to. Typical subjects include animals (eg, mice, rats, rabbits, non-human primates, and humans; insects; helminths; mammals such as others) and plants. In some embodiments, the subject is suffering from a disease, disorder, and / or symptom, and / or is susceptible to these.

Substantial: As used herein, the term "substantial" refers to a qualitative condition that indicates the extent or extent of all or almost all of the features or characteristics of interest. Those skilled in the art of biological technology will appreciate that biological and chemical phenomena rarely avoid completion and / or completion or achievement or absolute consequences. Therefore, the term "substantial" is used herein to capture the potential lack of integrity inherent in many biological and / or chemical phenomena.

Affected: An individual "affected" with a disease, disorder, and / or symptom has been diagnosed and / or indicates one or more symptoms of the disease, disorder, and / or symptom.

Predisposed to (disease): Diseases, disorders, and / or symptoms Individuals who are "prone to" are at higher risk of developing the disease, disability, and / or symptoms than members of the general public. In some embodiments, an individual susceptible to a disease, disorder, and / or symptom may not be diagnosed with the disease, disorder, and / or symptom. In some embodiments, an individual susceptible to a disease, disorder, and / or symptom may exhibit symptoms of the disease, disorder, and / or symptom. In some embodiments, an individual susceptible to a disease, disorder, and / or symptom may not exhibit symptoms of the disease, disorder, and / or symptom. In some embodiments, an individual susceptible to a disease, disorder, and / or symptom will develop the disease, disorder, and / or symptom. In some embodiments, an individual susceptible to a disease, disorder, and / or symptom will not develop the disease, disorder, and / or symptom.

Systemic: When used herein, the phrases "systemic administration," "systemic administration," "peripheral administration," and "peripheral administration" are the recipes. It has the meaning understood in the art to represent the administration of a compound or composition to enter the whole body of the ent.

Tautomer: As used herein, the phrase "tautomer" is used to describe an organic compound of a different isomer that is easily convertible. Tautomers can be characterized by formal transfer of hydrogen atoms or protons that occurs at the same time as the conversion of single and adjacent double bonds. In some embodiments, the tautomer can result from proton tautomerism (ie, proton rearrangement). In some embodiments, the tautomer can result from valence tautomerism (ie, rapid rearrangement of bound electrons). Such tautomers shall be included within the scope of the present invention. In some embodiments, tautomers of the compounds are in equilibrium that is mobile to each other so that attempts to synthesize separate substances result in a mixture. In some embodiments, the tautomer of the compound is a separable and separable compound. In some embodiments of the invention, the chemical composition may be provided as a pure synthesis of a single tautomer of the compound, or one containing it. In some embodiments of the invention, the chemical composition may be provided as a mixture of two or more tautomers of the compound. In certain embodiments, the mixture comprises the same amount of different tautomers; in certain embodiments, the mixture comprises at least two tautomers of the compound in different amounts. In some embodiments of the invention, the chemical composition may comprise all tautomers of the compound. In some embodiments of the invention, the chemical composition may contain less than all tautomers of the compound. In some embodiments of the invention, the chemical composition may comprise one or more tautomers of the compound in an amount that changes over time as a result of reciprocal conversion. In some embodiments of the invention, the tautomer is keto-enol tautomer. Those skilled in the field of chemical technology "capture" (ie, chemically modify) keto-enol tectonicity with any suitable reagent known in the field of chemical technology to retain the "enol" form. Thus, one or more suitable techniques well known in the art can be used to obtain enol derivatives that can be subsequently isolated. Unless otherwise indicated, the invention includes all tautomers of related compounds, whether in pure form or in mixtures with each other.

Therapeutic Agents: As used herein, the term "therapeutic agent" is any that, when administered to a subject, elicits a therapeutic effect and / or a desired biological and / or pharmacological effect. Represents a drug. In some embodiments, the therapeutic agent alleviates, remits, releases, suppresses, prevents, delays onset, reduces severity, and / or reduces one or more signs or features of the disease, disorder, and / or symptoms. Any substance that can be used to reduce the incidence.

Therapeutic Effective Amount: As used herein, the term "therapeutic effective amount" is a substance that elicits a desired biological response when administered as part of a therapeutic regimen (eg, a therapeutic agent, etc.). Means the amount of composition and / or formulation). In some embodiments, a therapeutically effective amount of the substance treats, diagnoses, or diagnoses the disease, disorder, and / or symptoms when administered to a subject suffering from or susceptible to the disease, disorder, and / or symptoms. Enough to prevent and / or delay the onset of the disease. As will be appreciated by those of skill in the art, the effective amount of a substance may vary depending on factors such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, and others. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or symptom may alleviate, ameliorate, release, alleviate, ameliorate, release, one or more signs or characteristics of the disease, disorder, and / or symptom. An amount that suppresses, prevents, delays the onset of the disease, reduces the severity of the disease, and / or reduces the incidence. In some embodiments, the therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver the therapeutically effective amount.

Treatment: As used herein, the terms "treat," "treat," or "treat" partially describe one or more signs or features of a disease, disorder, and / or symptom. Represents any method used to alleviate, remit, release, suppress, prevent, delay onset, reduce severity, and / or reduce the incidence, either completely or completely. Treatment can be administered to subjects who show no signs of illness, disability, and / or symptoms. In some embodiments, for the purpose of reducing the risk of developing a pathology associated with, for example, a disease, disorder, and / or symptom, treatment is directed to a subject showing only the initial signs of the disease, disorder, and / or symptom. Can be administered.

Unsaturation: As used herein, the term "unsaturated" means that a moiety has one or more unsaturated units.

Unit Dose: As used herein, the expression "unit dose" refers to an amount administered as a single dose of a pharmaceutical composition and / or in physically separate units. In many embodiments, the unit dose comprises a predetermined amount of active agent. In some embodiments, the unit dose comprises a whole single dose of the agent. In some embodiments, one or more unit doses are administered to achieve a total single dose. In some embodiments, administration of multiple unit doses is expected to be necessary or necessary to achieve the intended effect. A unit dose is, for example, a volume of liquid comprising a predetermined amount of one or more therapeutic agents, a predetermined amount of solid form, a sustained release formulation or a drug delivery device containing a predetermined amount of one or more therapeutic agents, and others. It can be (eg, an acceptable carrier). It will be appreciated that unit doses may be present in formulations containing any of the various ingredients in addition to the therapeutic agent. For example, acceptable carriers (eg, pharmaceutically acceptable carriers), diluents, stabilizers, buffers, preservatives, etc. may be included, as described below. In many embodiments, it is possible that the appropriate total daily dose of a particular therapeutic agent may include one or more unit doses, eg, be determined by the attending physician within sound medical judgment. It will be understood by those skilled in the art. In some embodiments, the specific effective dose level for any particular subject or organism is the disorder being treated and the severity of the disorder; the activity of the specific active compound used; Specific composition; subject's age, weight, health status, gender and diet; frequency of administration and excretion rate of specific compound used; duration of treatment; agent used in combination with or simultaneously with the specific compound used And / or additional therapies, and may depend on a variety of factors, including similar factors well known in the medical field.

Wild type: As used herein, the term "wild type" refers to a "normal" (mutant, sick, altered, as opposed to others) state or context. Representing an entity with structure and / or activity that is actually seen, has a meaning understood in the art. One of skill in the art will recognize that wild-type genes and polypeptides often exist in several different forms (eg, alleles).

Nucleic Acid: The term "nucleic acid" includes any nucleotide, its analog, and its polymer. As used herein, the term "polynucleotide" refers to a polymerization form of nucleotides of any length, ribonucleotide (RNA) or deoxyribonucleotide (DNA). These terms refer to the primary structure of the molecule and thus include double-stranded and single-stranded DNA, and double-stranded and single-stranded RNA. These terms, as equivalents, include, but are not limited to, nucleotide analogs such as methylated, protected and / or capped nucleotides or polynucleotides and RNA or DNA analogs synthesized from modified polynucleotides. include. The term is poly or oligoribonucleotide (RNA) and poly or oligodeoxyribonucleotide (DNA); N-glycosides of nucleobases and / or modified nucleobases or RNA or DNA derived from C-glycosides; saccharides and / or Nucleic acids derived from modified saccharides; and nucleic acids derived from phosphate cross-linking and / or modified phosphorus atom cross-linking (also referred to herein as "internucleotide linkage"). The term includes nucleic acids comprising any combination of nucleobases, modified nucleobases, saccharides, modified saccharides, phosphate crosslinks or modified phosphorus atom crosslinks. Examples include, but are not limited to, nucleic acids containing a ribose moiety, nucleic acids comprising a deoxyribose moiety, nucleic acids comprising both a ribose moiety and a deoxyribose moiety, and nucleic acids comprising a ribose moiety and a modified ribose moiety. The prefix poly represents a nucleic acid containing 2 to about 10,000 nucleotide monomer units, and the prefix oligo represents 2 to about 200 nucleotide monomer units.

Nucleotides: As used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide consisting of a heterocyclic base, a sugar, and one or more phosphate groups or phosphorus-containing internucleotide bonds. Natural bases (guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)) are purine or pyrimidine derivatives, but also include natural and unnatural base analogs. Should be understood. Natural sugars are pentose (pentose) deoxyribose (forming DNA) or ribose (forming RNA), but it should be understood that they also include natural and unnatural base analogs. Nucleotides bind via internucleotide bonds to produce nucleic acids, or polynucleotides. Many internucleotide bonds are well known in the art (such as, but not limited to, phosphoric acid, phosphorothioate, borane phosphate, etc.). Artificial nucleic acids include PNAs (peptide nucleic acids), phosphotriesters, phosphorothionates, H-phosphonic acid esters, amide subphosphates, boranophosphates, methylphonsphonic acid esters, and phosphonoacetic acid. Examples include esters, thiophosphonoacetate esters and other variants of the phosphate skeleton of natural nucleic acids such as those described herein.

Nucleoside: The term "nucleoside" refers to the portion of a nucleobase or modified nucleobase that is covalently attached to a sugar or modified sugar.

Sugar: The word "sugar" refers to closed and / or open monosaccharides. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexopyranose moieties. As used herein, the term also includes structural analogs in which the polymer is used in place of conventional sugar molecules such as nucleic acid analogs, glycols that form the backbone of glycol nucleic acids (“GNA”).

Modified sugar: The term "modified sugar" refers to a portion that can replace sugar. The modified sugar mimics spatial arrangement, electronic state, or some other physicochemical property of the sugar.

Nucleobase: The term "nucleobase" refers to a nucleic acid moiety associated with a hydrogen bond that binds one nucleic acid strand to another complementary strand in a sequence-specific manner. Most natural nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the natural nucleobase is modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the natural nucleobase is methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the nucleobase is a "modified nucleobase", eg, a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). .. In some embodiments, the modified nucleobase is methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the modified nucleobase mimics spatial arrangement, electronic state, or some other physicochemical property of the nucleobase, complementing one nucleobase with another in a sequence-specific manner. It retains the properties of hydrogen bonds that bind to chains. In some embodiments, the modified nucleobase is the five all-natural bases (uracil, thymine, adenine, cytosine) with virtually no effect on melting behavior, recognition by intercellular enzymes or activity of the oligonucleotide double strand. , Or guanine).

Chiral Ligand: The term "chiral ligand" or "chiral aid" is chiral and refers to a portion that can be incorporated into a reactant such that the reaction can be carried out with specific stereoselectivity.

Condensation Reagent: In a condensation reaction, the term "condensation reagent" refers to a reagent that activates a less reactive site and makes it more sensitive to action with another reagent. In some embodiments, such another reagent is a nucleophile.

Block group: The term "block group" refers to a group that shields the reactivity of a functional group. The functional group can subsequently remove the shield by removing the blocking group. In some embodiments, the blocking group is a protecting group.

Part: The word "part" refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized as chemical entities incorporated or added into the molecule.

Solid Carrier: The term "solid carrier" refers to any carrier that allows the synthesis of nucleic acids. In some embodiments, the term refers to a glass or polymer that is insoluble in the medium used in the reaction step of performing nucleic acid synthesis and inducing to introduce reactive groups. In some embodiments, the solid carrier is highly crosslinked polystyrene (HCP) or controlled pore glass (CPG). In some embodiments, the solid carrier is controlled pore glass (CPG). In some embodiments, the solid carrier is a composite carrier of controlled pore glass (CPG) and highly crosslinked polystyrene (HCP).

Binding moiety: The term "binding moiety" refers to any moiety that may be located between the terminal nucleotide and the solid carrier or between the terminal nucleoside and another nucleoside, nucleotide or nucleic acid.

DNA molecule: The term "DNA molecule" refers to its single-stranded or double-helix deoxyribonucleotide (adenine, guanine, thymine, or cytosine) polymerized form. The term refers only to the primary and secondary structure of the molecule and is not limited to any particular tertiary form. Thus, the term includes, among other things, linear DNA molecules (eg, restriction enzyme fragments), viruses, plasmids, and double-stranded DNA found in chromosomes. In discussing the specific double-stranded DNA molecular structure, the sequence follows the convention of giving only sequences in the 5'to 3'direction along the DNA of the non-transcribed strand (ie, a strand having a sequence homologous to the mRNA). It may be described in the specification.

Code sequence: DNA A "coding sequence" or "coding region" is a double-stranded DNA that is transcribed and translated into an in vivo polypeptide when placed under the control of an appropriate expression control sequence. The coding sequence boundaries (“open reading frame” or “ORF”) are determined by the start codon at the 5'(amino) end and the translation stop codon at the 3'(carboxylic acid) end. Coding sequences include, but are not limited to, prokaryotic sequences, cDNAs from prokaryotic mRNAs, genomic DNA sequences from prokaryotic (eg, mammalian) DNA, and synthetic DNA sequences. Polyadenylation signals and transcription termination sequences are usually located on the 3'side of the coding sequence, the term "non-coding sequence" or "non-coding region" is a region of the polynucleotide sequence that is not translated into amino acids (eg, 5). Represents'and 3'untranslated regions).

Reading frame: The term "reading frame" refers to one of three possible reading frames in each direction of a double-stranded DNA molecule. The reading frame used determines which codon is used to encode an amino acid in the coding sequence of the DNA molecule.

Antisense: As used herein, an "antisense" nucleic acid molecule is, for example, a "sense" complementary to the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence, or complementary to a gene coding strand. Includes a nucleic acid sequence complementary to the nucleic acid coding protein. Thus, the antisense nucleic acid molecule may associate with the sense nucleic acid molecule via hydrogen bonds.

Fluctuation position: As used herein, "fluctuation position" represents the third position of a codon. In some embodiments, mutations in DNA molecules within codon fluctuation positions result in silent or conservative mutations at the amino acid level. For example, there are four codons encoding glycine, namely GGU, GGC, GGA and GGG, so mutations of a nucleotide at any fluctuation position to another nucleotide selected from A, U, C and G , Does not result in changes at the amino acid level of the encoded protein and is therefore a silent substitution.

Silent Substitution: A "silent substitution" or "silent mutation" is one in which the nucleotides within a codon are altered but the amino acid residues encoded by the codon are not altered. Examples include mutations in the first position of a particular codon, such as the codon "CGG" that encodes Arg, as well as in the third position of the codon, even when mutated to AGG.

Gene: As used herein, the terms "gene," "recombinant gene," and "gene construct" refer to a DNA molecule, or part of a DNA molecule, that encodes a protein or portion thereof. The DNA molecule may comprise an open reading frame encoding the protein (as an exon sequence) and may further comprise an intron sequence. As used herein, the term "intron" refers to a DNA sequence that is present in a given gene that is not translated into a protein, and in some cases, but in some cases, between exons. As is well known in the art, a gene operably binds to (or may contain) one or more promoters, enhancers, repressors and / or other regulatory sequences that regulate the activity or expression of the gene. That can be desired.

Complementary DNA: As used herein, "complementary DNA" or "cDNA" comprises a recombinant polynucleotide synthesized by reverse transcription of an mRNA from which an intervening sequence (intron) is removed.

Homology: "homology" or "identity" or "similarity" represents sequence similarity between two nucleic acid molecules. Homology and identity can each be determined by comparing the position of each sequence that can be placed for comparison purposes. When the same position in the compared sequence is occupied by the same base, then the molecule is identical at that position; the same site is the same or similar nucleic acid residue (eg, steric and / or). When occupied by (similar in electronic state), the molecule can then be referred to as homologous (similar) at that position. The homology / similarity or percentage of identity representation represents a function of the number of identical or similar nucleic acids at the positions shared by the sequences being compared. An "irrelevant" or "non-homologous" sequence has less than 40% identity, less than 35% identity, less than 30% identity, or less than 25% identity with the sequences described herein. share it. When comparing two sequences, the lack of residues (amino acids or nucleic acids) or the presence of extra residues also reduces the identity and homology / similarity.

In some embodiments, the term "homology" describes a mathematically based comparison of sequence similarity used for genes that are identical to similar functions or motifs. The nucleic acid sequences described herein can be used, for example, as "query sequences" to perform a search against a public database to identify other family members, related sequences or homologues. In some embodiments, such a search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. In some embodiments, the BLAST nucleotide search can be performed with the NBLAST program, score = 100, word length = 12 to obtain a nucleotide sequence homologous to the nucleic acid molecule of the invention. In some embodiments, gap BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402 to obtain gap alignment for comparative purposes. When utilizing BLAST and Gap BLAST programs, the default parameters of their respective programs (eg, XBLAST and BLAST) may be used (see www.ncbi.nlm.nih.gov).

Identity: As used herein, "identity" means more than one sequence when aligning the sequences to maximize sequence matching, i.e., taking into account gaps and insertions. Means the percentage of identical nucleotide residues at the corresponding positions in the sequence. Identity can be easily calculated by known methods, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic. Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press , 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48 : 1073 (1988), but not limited to. The method of determining identity is designed to provide maximum matching between test sequences. In addition, the method of determining identity is coded in publicly available computer programs. Computer programming methods for determining the identity between two sequences include the GCG Program Package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASTP, BLASTN, and FASTA ( Altschul, SF et al., J. Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)), but not limited to. The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)). The well-known Smith Waterman algorithm can also be used for identity determination.

Non-homologous: The "non-homologous" region of a DNA sequence is an identifiable segment of DNA within a larger DNA sequence that is not found at all in relation to the larger sequence. Thus, when a non-homologous region encodes a mammalian gene, the gene may normally be on the DNA side not adjacent to the mammalian genomic DNA in the source organism's genome. Another example of a non-homologous coding sequence is a sequence in which the coding sequence itself is not found at all (eg, a cDNA containing an intron or synthetic sequence whose genomic coding sequence has a codon or motif different from that of the unmodified gene). Allelic or spontaneous mutation events do not give rise to non-homologous regions of DNA as defined herein.

Nucleotide sequence mutations: The term "nucleotide sequence mutation" refers to pyrimidines (cytidines (C) or thymidines (T)) being replaced by another pyrimidine, or purines (adenosine (A) or guanosine (G)). )) Represents a change in the base in the DNA sequence that is replaced by another purine.

Base-converted mutations: The term "base-converted mutations" refers to pyrimidines (cytidines (C) or thymidines (T)) being replaced by purines or purines (adenosine (A) or guanosine (G)). Represents a change in the base in the DNA sequence that is replaced by pyrimidine.

Oligonucleotides: The term "oligonucleotide" refers to nucleobases, modified nucleic acid bases, sugars, modified sugars, phosphate crosslinks, or modified phosphorus atomic crosslinks (in the present specification, further defined herein as "internucleotide linkage". Represents a polymer or oligomer of a nucleotide monomer containing any combination of).

Oligonucleotides can be single-stranded or double-stranded. As used herein, the term "oligonucleotide strand" includes single-stranded oligonucleotides. A single-stranded oligonucleotide can have a double-stranded region, and a double-stranded oligonucleotide can have a single-stranded region. Examples of oligonucleotides are self-replications such as structural genes, genes containing regulatory and termination regions, viral or plasmid DNA, single-stranded and double-stranded siRNAs and other RNA interfering agents (RNAi or iRNA agents). System, shRNA, antisense oligonucleotide, ribozyme, microRNA, microRNA mimic, supermir (supermir), aptamer, antimir (antimir), antagomir (antagomir), Ul adapter, triple-strand-forming oligonucleotide, guanine tetra Heavy chain oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides include, but are not limited to.

Double-stranded and single-stranded oligonucleotides that are effective in inducing RNA interference are also referred to herein as siRNA, RNAi agents, or iRNA agents. In some embodiments, these RNA interference-inducing oligonucleotides are associated with a cytoplasmic multiprotein complex known as RNAi-induced silencing complex (RISC). In many embodiments, single-stranded and double-stranded RNAi agents are due to the fact that they enter the RISC mechanism and produce smaller oligonucleotides that can be involved in RISC-mediated cleavage of the target sequence, eg, the target mRNA. Long enough to be cleaved by an endogenous molecule, eg, a dicer.

The oligonucleotides of the present invention can be of various lengths. In certain embodiments, the oligonucleotide can range from about 2 to about 200 nucleotides in length. In various related embodiments, single-stranded, double-stranded, and triple-stranded oligonucleotides are about 4 to about 10 nucleotides, about 10 to about 50 nucleotides, about 20 to about 50 nucleotides, and about 15 to about. It can range in length from 30 nucleotides, about 20 to about 30 nucleotides. In some embodiments, the oligonucleotide is about 9 to about 39 nucleotides in length. In some embodiments, the oligonucleotide is at least 4 nucleotides in length. In some embodiments, the oligonucleotide is at least 5 nucleotides in length. In some embodiments, the oligonucleotide is at least 6 nucleotides in length. In some embodiments, the oligonucleotide is at least 7 nucleotides in length. In some embodiments, the oligonucleotide is at least 8 nucleotides in length. In some embodiments, the oligonucleotide is at least 9 nucleotides in length. In some embodiments, the oligonucleotide is at least 10 nucleotides in length. In some embodiments, the oligonucleotide is at least 11 nucleotides in length. In some embodiments, the oligonucleotide is at least 12 nucleotides in length. In some embodiments, the oligonucleotide is at least 15 nucleotides in length. In some embodiments, the oligonucleotide is at least 20 nucleotides in length. In some embodiments, the oligonucleotide is at least 25 nucleotides in length. In some embodiments, the oligonucleotide is at least 30 nucleotides in length. In some embodiments, the oligonucleotide is a double-stranded complementary strand with a length of at least 18 nucleotides. In some embodiments, the oligonucleotide is a double-stranded complementary strand with a length of at least 21 nucleotides.

または修飾ホスホロチオエートトリエステル結合である。該ヌクレオチド間結合が、該結合の酸または塩基部分の存在により、所与のｐＨにおいて、アニオンまたはカチオンとして存在し得ることを、当業者は理解している。 Internucleotide Binding: As used herein, the phrase "internucleotide binding" generally refers to a phosphorus-containing bond between nucleotide units of an oligonucleotide, as described above and herein, "internucleotide binding." And "phosphorus atom cross-linking" are synonymous. In some embodiments, the internucleotide bond is a phosphodiester bond found in native DNA and RNA molecules. In some embodiments, the internucleotide bond is a "modified internucleotide bond" in which each oxygen atom of the phosphodiester bond is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, such organic or inorganic moieties are = S, = Se, = NR', -SR', -SeR', -N (R') ₂ , B (R') ₃ , -S. -, -Se-, and -N (R')-(in the formula, each R'is independently defined and described below), but is not limited thereto. In some embodiments, the internucleotide bond is a phosphodiester bond, a phosphodiester bond.

Alternatively, it is a modified phosphorothioate triester bond. Those skilled in the art will appreciate that the internucleotide bond may be present as an anion or cation at a given pH due to the presence of the acid or base portion of the bond.

Unless otherwise specified, when used with oligonucleotide sequences, each s, s1, s2, s3, s4, s5, s6 and s7 independently bind to the following modified nucleotides shown in Table 1 below. Represents.

Table 1. An example of modified internucleotide binding

）
；および２）ＣとＧ間の

の構造を有するホスホロチオエートトリエステルヌクレオチド間結合を有する。特に指定しない限り、オリゴヌクレオチド配列に先行するＲｐ／Ｓｐの表記は、該オリゴヌクレオチド配列の順次５’から３’の該ヌクレオチド間結合のキラル結合リン原子の立体配置を言い表す。例えば、（Ｒｐ，Ｓｐ）－ＡＴｓＣｓ１ＧＡでは、ＴとＣ間の「ｓ」結合のリンは、Ｒｐ立体配置を有し、ＣとＧ間の「ｓ１」結合のリンは、Ｓｐ立体配置を有する。いくつかの実施形態では、「全（Ｒｐ）」または「全（Ｓｐ）」は、オリゴヌクレオチドの全キラル結合リン原子が、それぞれ、同じＲｐまたはＳｐ立体配置を有することを示すために使用される。例えば、全（Ｒｐ）－ＧｓＣｓＣｓＴｓＣｓＡｓＧｓＴｓＣｓＴｓＧｓＣｓＴｓＴｓＣｓＧｓＣｓＡｓＣｓＣは、該オリゴヌクレオチドの全ての該キラル結合リン原子が、Ｒｐ立体配置を有することを示す；全（Ｓｐ）－ＧｓＣｓＣｓＴｓＣｓＡｓＧｓＴｓＣｓＴｓＧｓＣｓＴｓＴｓＣｓＧｓＣｓＡｓＣｓＣは、該オリゴヌクレオチドの全ての該キラル結合リン原子が、Ｓｐ立体配置を有することを示す。 For example, (Rp, Sp) -ATsCs1GA is 1) a phosphorothioate nucleotide-to-nucleotide binding between T and C (

)
And 2) between C and G

It has a phosphorothioate triester nucleotide-to-nucleotide bond having the structure of. Unless otherwise specified, the Rp / Sp notation preceding the oligonucleotide sequence refers to the configuration of the chiral-bound phosphorus atom of the internucleotide bond sequentially 5'to 3'of the oligonucleotide sequence. For example, in (Rp, Sp) -ATsCs1GA, the "s" -bound phosphorus between T and C has an Rp configuration and the "s1" -bound phosphorus between C and G has a Sp configuration. In some embodiments, "whole (Rp)" or "whole (Sp)" is used to indicate that all chirally bound phosphorus atoms of an oligonucleotide have the same Rp or Sp configuration, respectively. .. For example, all (Rp) -GsCsCsTsCsGsTsCsTsGsCsTsTsCsGsCsCsC indicates that all the chirally bound phosphorus atoms of the oligonucleotide have an Rp configuration; It is shown that the atom has a Sp configuration.

Oligonucleotide type: As used herein, the phrase "oligonucleotide type" refers to a particular base sequence, skeletal binding pattern (ie, internucleotide binding pattern, eg, phosphoric acid, phosphorothioate, etc.). Used to define oligonucleotides with skeletal chiral center patterns (ie, bound phosphorus stereochemical patterns (Rp / Sp)), and skeletal phosphorus modification patterns (eg, patterns of the "-XLR ¹ " group of formula I). To. Commonly described "type" oligonucleotides are structurally identical to each other.

Those skilled in the art are designed such that each nucleotide unit of an oligonucleotide chain has a particular steric chemistry and / or a particular modification and / or a particular base and / or a particular sugar in the bound phosphorus. It will be appreciated that the synthetic methods of the invention provide some control during the synthesis of oligonucleotide chains, and / or as can be selected in advance. In some embodiments, oligonucleotide chains are designed and / or determined to have a particular combination of modifications in the bound phosphorus. In some embodiments, oligonucleotide chains are designed and / or selected because they have a particular combination of bases. In some embodiments, oligonucleotide chains are designed and / or selected to have one or more of the above structural features of a particular combination. The present invention provides a composition comprising or consisting of a plurality of oligonucleotide molecules (eg, a chirally controlled oligonucleotide composition). In some embodiments, all such molecules are of the same type (ie, structurally identical to each other). However, in many embodiments, the provided composition usually comprises a plurality of different types of oligonucleotides in predetermined relative amounts.

Chiral control: As used herein, "chiral control" refers to the ability to control the stereochemical notation of each chiral-bound phosphorus within an oligonucleotide chain. The phrase "chiral-controlled oligonucleotide" refers to an oligonucleotide that is present in a single diastereoisomer with respect to the chirally bound phosphorus.

Chiral Controlled Oligonucleotide Compositions: As used herein, the phrase "chiral controlled oligonucleotide composition" refers to an oligonucleotide composition comprising a given level of individual oligonucleotide types. For example, in some embodiments, the chiral controlled oligonucleotide composition comprises one oligonucleotide type. In some embodiments, the chiral controlled oligonucleotide composition comprises a mixture of multiple oligonucleotide types. Illustrative chiral controlled oligonucleotide compositions are further described herein.

Chiral Pure: As used herein, the phrase "chiral pure" is used to describe a chiral-controlled oligonucleotide composition in which all oligonucleotides are present in a single diastereoisomer with respect to the bound phosphorus.

Chiral Uniformity: As used herein, the phrase "chiral uniform" is used to describe an oligonucleotide molecule or type in which all nucleotide units have the same stereochemistry in the bound phosphorus. For example, oligonucleotides having Rp stereochemistry, all of which have their nucleotide units all bound to phosphorus, are chirally uniform. Similarly, oligonucleotides having Sp stereochemistry, all of which have their nucleotide units all bound to phosphorus, are chirally uniform.

Predetermined: Predetermined means to be deliberately selected, for example, as an opposite of random occurrence or achievement. Those skilled in the art will read this specification to allow the invention to select a particular oligonucleotide type for dispensing and / or encapsulation of the provided composition so that the provided composition is dispensed. , Optionally, will be understood to provide new and amazing techniques that further enable controlled dispensing of the selected specific type, with the selected specific relative amount, exactly. Such provided compositions are the "predetermined" ones described herein. A composition that may contain a particular individual oligonucleotide type is not a "predetermined" composition, as they happened to be made through a process in which the intentional creation of a particular oligonucleotide type could not be controlled. In some embodiments, a given composition can be deliberately replicated (eg, through a controlled process iteration).

Bound Phosphorus: As defined herein, the phrase "bound phosphorus" means that the particular phosphorus atom represented is present in internucleotide bonds and that phosphorus atom occurs in native DNA and RNA. It is used to indicate that it corresponds to the phosphorus atom of an internucleotide phosphodiester. In some embodiments, the bound phosphorus atom is in a modified internucleotide bond and each oxygen atom of the phosphodiester bond is optionally and independently substituted by an organic or inorganic moiety. In some embodiments, the bound phosphorus atom is P ^* of formula I. In some embodiments, the bound phosphorus atom is chiral. In some embodiments, the chiral-bonded phosphorus atom is P ^* of formula I.

P-modification: As used herein, the term "P-modification" refers to any modification in bound phosphorus other than stereochemical modification. In some embodiments, the P modification comprises the addition, substitution, or removal of a covalently bound suspension moiety to the bound phosphorus. In some embodiments, the "P-modification" is -X-L-R ¹ (where X, L and R ¹ are independently defined and described herein and below. ).

Brockmer: As used herein, the term "Brockmer" means that the patterns of structural features that characterize each individual nucleotide unit share common structural features in the internucleotide phosphorus binding. Represents an oligonucleotide chain characterized by the presence of at least two contiguous nucleotide units. Common structural features mean common stereochemistry in the bound phosphorus or common modifications in the bound phosphorus. In some embodiments, the at least two contiguous nucleotide units that share common structural features in the internucleotide phosphorus binding are referred to as "blocks".

In some embodiments, the blockmer is a "stereoblockmer", eg, at least two contiguous nucleotide units have the same stereochemistry in the bound phosphorus. Such at least two contiguous nucleotide units form a "stereo blocker". For example, (Rp, Sp) -ATsCs1GA is a stereoblockmer because at least two contiguous nucleotide units, Ts and Cs1, have the same stereochemistry at bound phosphorus (both Sp). In the same oligonucleotide, (Rp, Sp) -ATsCs1 forms a block, which is a steric block.

In some embodiments, the blockmer is a "P-modified blockmer", eg, at least two contiguous nucleotide units have the same modification in the bound phosphorus. Such at least two contiguous nucleotide units form a "P-modified block". For example, (Rp, Sp) -ATsCsGA is a P-modified blockmer because at least two contiguous nucleotide units, Ts and Cs, have the same P modification (ie, both are phosphodiester diesters). In the same oligonucleotide of (Rp, Sp) -ATsCsGA, TsCs form a block, which is a P-modified block.

In some embodiments, the blockmer is a "bound blockmer", for example, at least two contiguous nucleotide units have the same stereochemistry and the same modification in the bound phosphorus. At least two contiguous nucleotide units form a "binding block". For example, (Rp, Rp) -ATsCsGA is a binding blocker because at least two contiguous nucleotide units, Ts and Cs, have the same stereochemistry (both Rp) and P modification (both phosphorothioates). In the same oligonucleotide of (Rp, Rp) -ATsCsGA, TsCs form a block and are a binding block.

In some embodiments, the blockmer independently comprises one or more blocks selected from a three-dimensional block, a P-modified block and a coupled block. In some embodiments, the blockmers are stereo blockmers for one block and / or P-modified blockmers for another block, and / or binding blockmers for yet another block. For example, (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp) -AAsTsCsGsAs1Ts1Cs1Gs1ATCG is a stereoblock for the three-dimensional block AsTsCsGsAs1 (all Rp in bound phosphorus) or Ts1Cs1Gs1 (all Sp in bound phosphorus). Modified block AsTsCsGs (total s binding) or As1Ts1Cs1Gs1 (total s1 binding) is a P-modified blocker, or binding block AsTsCsGs (total Rp and total s binding in bound phosphorus) or Ts1Cs1Gs1 (total Sp and total s1 binding in bound phosphorus). ) Is a combined blocker.

Altmer: As used herein, the term "altmer" is a structural feature pattern that characterizes each individual nucleotide unit and shares certain structural features in the internucleotide phosphorus binding. Represents an oligonucleotide chain characterized by the absence of two contiguous nucleotide units in the oligonucleotide chain. In some embodiments, the altmer is designed to include a repeating pattern. In some embodiments, the altmer is designed so that it does not contain a repeating pattern.

In some embodiments, the altomer is a "stereo altomer", eg, there are no two contiguous nucleotide units with the same stereochemistry in the bound phosphorus. For example, (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp) -GsCsCsTsCsAsGsTsCsTsGsCsTsCsCsCs.

In some embodiments, the altomer is a "P-modified altomer", eg, there are no two consecutive nucleotide units with the same modification in the bound phosphorus. For example, total (Sp) CAs1GsT, where each bound phosphorus has a different P modification than the others.

In some embodiments, the altomer is a "bound altomer", eg, there are no two consecutive nucleotide units with the same stereochemistry or the same modification in the bound phosphorus. For example, (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp) -GsCs1CsTs1CsAs1GsTs1CsTs1GsCs1TsCs2.

Unimer: As used herein, the term "unimer" characterizes each individual nucleotide unit in its structural feature pattern, where all nucleotide units within the strand are at least one in the internucleotide phosphorus bond. Represents an oligonucleotide chain that shares common structural features. Common structural features mean common stereochemistry in the bound phosphorus or common modifications in the bound phosphorus.

In some embodiments, the unimmer is a "stereo unimmer", eg, all nucleotide units have the same stereochemistry in the bound phosphorus. For example, all (Sp) -CsAs1GsT, all of which have Sp phosphorus.

In some embodiments, the unimmer is a "P-modified unimmer", eg, all nucleotide units have the same modification in the bound phosphorus. For example, all of the internucleotide bonds are phosphodiester bonds (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp. ) -GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC.

In some embodiments, the unimmer is a "bound unimmer", for example, all nucleotide units have the same stereochemistry and the same modifications in the bound phosphorus. For example, all (Sp) -GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsCsC, where all of the creatide bonds are phosphodiesters with Sp-bound phosphorus.

Gapmer: As used herein, the term "gapmer" means that at least one internucleotide phosphorus bond in an oligonucleotide chain is found in a phosphodiester bond, eg, natural DNA or RNA. Represents an oligonucleotide chain characterized by being. In some embodiments, the internucleotide phosphorus bond of one or more of the oligonucleotide chains is a phosphodiester bond, such as that found in native DNA or RNA. For example, the total (Sp) -CAs1GsT where the internucleotide bond between C and A is a phosphate diester bond.

Skipmer: As used herein, the term "skipmer" means that every other nucleotide-to-nucleotide phosphorus bond in the oligonucleotide chain is found in a phosphodiester bond, eg, natural DNA or RNA. Represents the type of gapmer, where every other internucleotide phosphodiester bond in the oligonucleotide chain is a modified internucleotide conjugation. For example, all (Sp) -AsTCs1GAs2TCs3G.

For the purposes of the present invention, chemical elements are specified according to the Periodic Table of the Elements on the Inner Cover, edited by CAS, Handbook of Chemistry and Physics, Edition 67, 1986-87.

The methods and structures described herein for compounds and compositions of the invention also apply to pharmaceutically acceptable acids or base adducts and all stereoisomers of these compounds and compositions.

Chiral controlled oligonucleotides have significantly different retention times on HPLC compared to stereorandom oligonucleotides. A: Crude chiral controlled oligonucleotide (oligonucleotide 101); C: Corresponding stereorandom oligonucleotide (oligonucleotide 118).

HPLC of chiral controlled oligonucleotides and stereorandom oligonucleotides. A: Oligonucleotide 101 (total Rp); B: Oligonucleotide 102 (total Sp); and C: Oligonucleotide 118 (stereo random).

Tm of chiral controlled oligonucleotides and stereorandom oligonucleotides.

Representative data: Melting curve analysis of single-unit replicating sequences from target and endogenous control pairs.

Representative data of compounds and IC ₅₀ .

Coarse (Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp) -d [ _{GsCsCsTsCsGsTsCsTsGsCsTsCsCs} -R, stereo blockmer and P-modified unimmer (s unimmer) HPLC.

Purification (Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp) -d [ _{GsCsCsTsCsGsTsCsTsGsCsTsCsCs} . -R, stereo blocker and P-modified unimmer (s unimmer) HPLC.

(Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, _Rp ) LCMS for R, stereo blockmer and P-modified unimmer (s unimmer).

Coarse (Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp) ) ₆ , Stereo blocker and P-modified unimmer (s unimmer)) HPLC.

Purification (Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp) ) ₆ , Stereo blocker and P-modified unimmer (s unimmer)) HPLC.

(Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp) -d [GsCsCsTsCsAsGsTsCsTsGsCsTsCsRSS _6. LCMS of stereo blocker and P-modified unimmer (s-unimer).

Coarse (Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp) d [GsCsCsTsCsAsGsTsCsTsGsCsTsCsRSCs! ₅ -RR, stereo blockmer and P-modified unimmer (s unimmer) HPLC.

Purification (Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp) d [GsCsCsTsCsAsGsTsCsTsGsCsTsCsRSCs. ₅ -RR, stereo blockmer and P-modified unimmer (s unimmer) HPLC.

(Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp) -d [GsCsCsTsCsAsGsTsCsTsGsCsTsCsRSS ₅ -RR, stereo blockmer and P-modified unimmer (s unimmer) LCMS.

Coarse (Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp) -d [5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (3R-5S-3R, Stereo Blocker) HPLC.

Purification (Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp) -d [5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (3R-5S-3R, Stereo Blocker) HPLC.

(Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp) -d [5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (3R-5S-3R, stereo blocker) LCMS.

HPLC of crude (Rp) -d [Cs3As3Gs3T] (P-modified unima- (s3 unima), stereo unimmer and bound unima-).

LCMS of all (Rp) -d [Cs3As3Gs3T] (P-modified unima- (s3 unima), stereo unimmer and coupled unima-).

HPLC of crude (Rp) -d [Cs2As2Gs2T] (P-modified unima- (s2 unima), stereo unimmer and coupled unima-).

LCMS of all (Rp) -d [Cs2As2Gs2T] (P-modified unima- (s2 unima), stereo unimmer and coupled unima-).

HPLC of crude (Sp) -d [Cs1AGs1T] (gapmer, stereo altomer, P-modified altomer and bound altomer).

LCMS of whole (Sp) -d [Cs1AGs1T] (gapmer, stereo altomer, P-modified altomer and bound altomer).

Coarse (Rp) -d [TsCs1AsT] (stereo unimmer, P-modified altomer and bound altomer).

LCMS of whole (Rp) -d [TsCs1AsT] (stereo unimmer, P-modified altomer and bound altomer).

An example oligonucleotide described in WO2012 / 030683 and contemplated by synthesis using the methods of the invention.

An example oligonucleotide described in WO2012 / 030683 and contemplated by synthesis using the methods of the invention.

An example oligonucleotide described in WO2012 / 030683 and contemplated by synthesis using the methods of the invention.

An example oligonucleotide described in WO2012 / 030683 and contemplated by synthesis using the methods of the invention.

An example oligonucleotide described in WO2012 / 030683 and contemplated by synthesis using the methods of the invention.

An exemplary linker according to WO2012 / 030683 for use in the method of the invention.

An exemplary linker according to WO2012 / 030683 for use in the method of the invention.

An exemplary linker according to WO2012 / 030683 for use in the method of the invention.

An exemplary linker according to WO2012 / 030683 for use in the method of the invention.

Crude DMT-treated oligonucleotides: ONT-75 (Panel A); ONT-80 (Panel B); ONT-77 (Panel C); ONT-81 (Panel D); ONT-87 (Panel E); ONT-88 ( Reversed phase HPLC of panel F); ONT-89 (panel G); ONT-82 (panel H); ONT-84 (panel I); ONT-85 (panel J); ONT-86 (panel K).

Oligonucleotides without purified DMT treatment: ONT-75 (panel A); ONT-80 (panel B); ONT-77 (panel C); ONT-81 (panel D); ONT-87 (panel E); ONT-88 (Panel F); ONT-89 (Panel G); ONT-82 (Panel H); ONT-84 (Panel I); ONT-85 (Panel J); ONT-86 (Panel K) reverse phase HPLC.

Oligonucleotides without purified DMT treatment: ONT-75, ONT-77, ONT-80, ONT-81, ONT-87, ONT-88, ONT-89, andONT-41 (panel A) overlays; ONT-75, ONT Overlay of reverse phase HPLC traces of -77, ONT-80, ONT-81, ONT-87, ONT-88, ONT-89, and ONT-41 (Panel B).

Oligonucleotides without purified DMT treatment: ONT-82, ONT-84, ONT-85, ONT-86, and ONT-83 (Panel A) reverse phase HPLC trace overlays; ONT-82, ONT-84, ONT-85. , ONT-86, and ONT-83 (Panel B) reverse phase HPLC trace overlay.

Tm overlays of chiral controlled oligonucleotides ONT-81, ONT-41, ONT-75, ONT-77, and ONT-80.

Serum human apolipoprotein B compared to PBS after intraperitoneal administration of 5 mg / kg stereoisomer or mipomersen to TON-41, ONT-75, ONT-80, ONT-77, and ONT-81 in huApoB mice. Illustration of the passage of time at the protein level. The down arrow indicates the dosing date.

Serum compared to PBS after intraperitoneal administration of 5 mg / kg stereoisomer or mipomelsen to mipomersen, "whole R" mipomersen, "whole S" mipomelsen, "RSR" mipomelsen, and "SRS" mipomelsen in huApoB mice Illustration of the time course of human apolipoprotein B protein levels. The down arrow indicates the dosing date.

Serum compared to PBS after intraperitoneal administration of 10 mg / kg mipomersen or mipomersen to mipomersen, "whole R" mipomersen, "whole S" mipomersen, "RSR" mipomelsen, and "SRS" mipomelsen in huApoB mice Illustration of the time course of human apolipoprotein B protein levels. The down arrow indicates the dosing date.

Time course of serum human apolipoprotein B protein levels compared to PBS after intraperitoneal administration of 5 mg / kg stereoisomer or mipomersen to mipomersen, ONT-87, ONT-88, and ONT-89 in huApoB mice. Illustrated. The down arrow indicates the dosing date.

Illustration of the time course of serum human apolipoprotein B protein levels compared to PBS after intraperitoneal administration of 10 mg / kg stereoisomers or mipomersen to TON-87, ONT-88, and ONT-89 in huApoB mice. The down arrow indicates the dosing date.

Illustration of residual PCSK-9 mRNA% after Hep3B treatment with siRNA double strand.

Illustration of residual PCSK-9 mRNA% after Hep3B treatment with siRNA double-stranded curve fitting.

..

Illustration of residual PCSK-9 mRNA% after HeLa treatment with siRNA double strand.

Illustration of residual PCSK-9 mRNA% after HeLa treatment with siRNA double-stranded curve fitting.

Illustration of residual PCSK-9 mRNA% after HeLa treatment with siRNA double strand containing 3 phosphorothioate stereocenters.

Illustration of residual PCSK-9 mRNA% after HeLa treatment with siRNA duplex containing 3 phosphorothioate stereocenter curve fitting.

Oligonucleotides without purified DMT treatment: overlay of reverse phase HPLC traces of ONT-108, ONT-109, and ONT-114.

Oligonucleotides without purified DMT treatment: overlay of reverse phase HPLC traces of ONT-106, ONT-107, and ONT-114.

Illustration of the time course of serum human apolipoprotein B protein levels compared to PBS after intraperitoneal administration of 10 mg / kg stereoisomer or mipomersen to huApoB mice. The down arrow indicates the dosing date.

Illustration of the time course of serum human apolipoprotein B protein levels compared to PBS after multiple intraperitoneal administrations of 5 mg / kg stereoisomers or mipomersen to huApoB mice (n = 3-4). The down arrow indicates the dosing date.

Serum human apolipoprotein B protein levels compared to PBS day 17 after intraperitoneal administration of 10 mg / kg stereoisomer (ONT-87, ONT-88 or ONT-89) or mipomersen to huApoB mice.

Serum human apolipoprotein B protein levels compared to PBS day 24 after intraperitoneal administration of 10 mg / kg stereoisomer (ONT-87, ONT-88 or ONT-89) or mipomersen to huApoB mice.

Serum human apolipoprotein B protein levels compared to PBS after administration of 10 mg / kg stereoisomers (ONT-41, ONT-87, ONT-88 or ONT-89) to huApoB mice.

Serum human apolipoprotein B protein levels compared to PBS after administration of 10 mg / kg stereoisomer (ONT-87, ONT-88 or ONT-89) to huApoB mice.

Plots of ion exchange HPLC quantitative analysis of svPDE digestion studies for oligonucleotides ONT-75, ONT-77, ONT-80, ONT-81, ONT-87, ONT-88, ONT-89 and ONT-41.

Oligonucleotide ONT-75 (whole (Rp))- Gs5mCs5mCsTs5mCs AsGsTs5mCsTsGs5mCsTsTs5mCs Gs5mCsAs5mCs5mC Ion exchange HPLC for enzyme digestion studies using nP1.

Oligonucleotide ONT-77 (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp ) -Gs5mCs5mCsTs5mCsAsGsTs5sCs5sCs5sCs5sCs Ion exchange HPLC for enzyme digestion studies using nP1 for -10S-4R).

Oligonucleotide ONT-80 (total (Sp))-Ion exchange HPLC for enzyme digestion studies using nP1 for Gs5mCs5mCsTs5mCs AsGsTs5mCsTsGs5mCsTsTs5mCs Gs5mCsAs5mCs5mC.

Oligonucleotide ONT-81 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp ) -Gs5mCs5mCsTs5mCsAsGsTs5sCs5sCs5sCs5sCs5s Ion exchange HPLC for enzyme digestion studies using nP1 for -10R-4S).

Oligonucleotide ONT-87 (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp ) -Gs5mCs5mCsTs5mCsAsGsTs5sCs5sCs5sCs5sCs -(SSR) 3-5R) ion exchange HPLC for enzyme digestion studies using _nP1 .

Oligonucleotide ONT-88 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp ) -Gs5mCs5mCsTs5mCsAsGsTs5sCs5sCs5sCs5sCs5s -Ion exchange HPLC for enzyme digestion studies using nP1 for (RRS) _3-5S ).

Oligonucleotide ONT-89 (Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp ) -Gs5mCs5mCsTs5mCsAsGsTs5sCs5sCs5sCs5s SR) Ion exchange HPLC for enzyme digestion studies using _nP1 for 9S).

Oligonucleotide ONT-41 (Diastereomix) -Ion exchange HPLC for enzyme digestion studies using nP1 for Gs5mCs5mCsTs5mCs AsGsTs5mCsTsGs 5mCsTsTs5mCs Gs5mCsAs5mCs5mC.

Stability comparison of the chiral pure oligonucleotides ONT-75 and ONT-77 with the stereorandom "parent" oligonucleotide ONT-41 (mipomersen) of the pre-incubated rat whole liver homogenate.

UPLC profile for oligonucleotide derivative production using 13b monomer.

UPLC profile for oligonucleotide derivative production using 27 monomers.

Mouse apolipoprotein B / GAPDH mRNA compared to simulated and untreated controls after transfection of primary cultured mouse hepatocytes with stereoisomers (ONT-82, ONT-83, ONT-84, ONT-85 or ONT-86). level.

Mouse apolipoprotein B / GAPDH mRNA levels compared to simulated and untreated controls after transfection of primary cultured mouse hepatocytes with stereoisomers (ONT-83, ONT-84, ONT-85 or ONT-86).

Synthetic oligonucleotides provide useful molecular tools for a wide variety of applications. For example, oligonucleotides are useful in therapeutic, diagnostic, research and novel nanomaterial applications. The use of native nucleic acids is limited by their susceptibility to (eg, unmodified DNA or RNA), eg, endo and exonucleases. Therefore, various synthetic equivalents have been developed to avoid these disadvantages. Synthetic equivalents include synthetic oligonucleotides containing skeletal modifications that make these molecules less prone to degradation. From a structural point of view, modification of such an polynucleotide to phosphate binding introduces chirality. It has been shown that certain properties of oligonucleotides can be influenced by the arrangement of phosphorus atoms that form the skeleton of the oligonucleotide. For example, in vitro studies have shown that the properties of antisense nucleotides, such as binding affinity, sequences that specifically bind to complementary RNA, and stability to nucleases, are particularly affected by skeletal chirality (eg, phosphorus). Arrangement of atoms). Accordingly, the present invention includes the recognition that there is a need for chirally controlled oligonucleotides containing phosphorus atom modified nucleic acids and their related compositions and methods. In some embodiments, the invention is structurally optimized and chiral controlled to exhibit certain desirable properties, such as increased stability and increased efficacy in in vitro and / or in vivo applications. Provide oligonucleotides.

Oligonucleotides in which one or two of the two non-bridged oxygen atoms of the phosphate of the internucleotide are replaced with different types of atoms or substituents are known to be useful as therapeutic agents. Investigations are underway to clarify the mechanism of enzymatic reactions. However, such oligonucleotides may exhibit undesired properties that make their use unusable in many applications (eg, susceptibility to degradation by nucleases, poor cell membrane permeability). Thus, various types of chemical modifications have been developed to improve properties and / or provide new functions.

Modified Oligonucleotide Structures As mentioned above, given the usefulness of oligonucleotide compositions in a variety of uses and conditions, those skilled in the art will compare them to native oligonucleotide molecules and utilize them, for example, for specific uses and conditions. Efforts have been made to develop modifications of oligonucleotide structures that may have favorable or desirable properties or properties. Such exemplary modifications are described below.

WO 2010/141471 (“Traversa I”) teaches the modification of different types of nucleic acid configurations modified to have a reduced net polyanionic charge. WO 2010/039543 (“Travera II”) teaches the composition and preparation of neutral polynucleotides (NNs) using reduced polyanionic charges. WO 2008/008676 (here, "Traversa III") describes the synthesis of SATE (Imbach-type) phosphate prodrugs. Traversa I, II and III do not disclose the chirally controlled oligonucleotides described in the present invention, their compositions, and methods of preparation using them.

WO 2010/072831 (“Girindus et al.”) Also teaches the modification of oligonucleotides. In particular, Gilindus et al. Disclose the use of sulfides to produce phosphorothioate triesters as prodrugs. Gilindus et al. Do not disclose the chirally controlled oligonucleotides described in the present invention, their compositions, and methods of preparation using them.

Similarly, WO 2004/085454 (“Avecia I”) teaches, for example, the preparation of phosphorothioate oligonucleotides through transient silylation of poly-H-phosphonate diesters. WO 2001/027126 (“Avecia II”) couples an H-phosphonate monomer to a solid-supported 5'-hydroxyl oligonucleotide and further converts the resulting H-phosphonate diester into a phosphorothioate triester. It teaches the process of solid phase synthesis of phosphodiester oligonucleotides that sulphurize. The disclosure of WO 2001/064702 (“Avecia III”) is similar to Avecia II and describes solid phase synthesis for a different solid support. Avecia I, II, and III do not disclose the chirally controlled oligonucleotides described in the present invention, their compositions, and methods of preparation using them.

WO 1997/006183 (“Chiron”) teaches oligonucleotides that have cation internucleotide bonds and contain asymmetric phosphorus, such as sterically pure amidates. Chiron teaches sterically pure oligonucleotides obtained by crystallizing with a mixture of diastereomers or a solution such as, for example, column chromatography. Chiron does not teach the chirally controlled oligonucleotides described in the present invention, their compositions, and methods of making them.

WO 2009/146123 (“Spring Bank I”) discloses compositions and methods for treating viral infections with substituted phosphate oligonucleotides and phosphorothioate triesters. WO 2007/070598 (“Spring Bank II”) teaches the synthesis of phosphotriester prodrugs and phosphorothioate prodrugs as antiviral nucleic acids. Spring Banks I and II do not disclose the chirally controlled oligonucleotides described in the present invention, their compositions, and methods of preparation using them.

European Patent No. 0779893 (“Hybridon”) teaches lipophilic prodrugs that incorporate many cells of antisense oligonucleotides, with Rp and Sp phosphorothioates and phosphorothioate triester dimers having different enzymatic stability. It was confirmed. Hybridon does not disclose the chirally controlled oligonucleotides described in the present invention, their compositions, and methods of preparation using them.

WO 1997/047673 (“Imbach I”) generally teaches Imbach “SATE” (S-acylthioethyl) prodrug oligonucleotide compositions and methods. Imbach I describes the preparation of certain prodrug oligonucleotides using, for example, bioreversible phosphotriester prodrugs and post-synthetic alkylation or phosphoramides containing prodrug groups. U.S. Pat. No. 6,124,445 (“Imbach II”) teaches modified antisense and chimeric prodrug oligonucleotides. Imbach I and II do not disclose the chirally controlled oligonucleotides described in the present invention, their compositions, and methods of preparation using them.

WO 2006/065751 (“Beaucage”) teaches about CpG oligonucleotide phosphorothioate prodrugs containing thermally unstable substituents (substituents introduced through phosphoramidite monomers) and their uses. There is. BEAUCAGE does not disclose the chirally controlled oligonucleotides described in the present invention, their compositions, and methods of preparation using them.

Takeshi Wada et al. Have developed a novel method for sterically controlled synthesis of P-chiral nucleic acids using chiral auxiliary (Japanese Patent No. 4348077, International Publication No. 2005/014609, International Publication No. 2005/92909). , And International Publication No. 2010/064146, referred to herein as "Wada I" in order). In particular, WO 2010/06414 (referred to herein as "Wada II") discloses a method for synthesizing a phosphate atom-modified nucleic acid in which the stereochemical arrangement in phosphorus is controlled. However, the Wada II method is limited in that it does not provide the individual P-modification of each chirally bound phosphate in a controlled and designed manner. That is, the method of binding the P-modification of Wada II, once made to the desired length, is mass-modified with the bound phosphate, eg, the desired phosphorothioate diester, phosphoramic acid or boranophosphate or another such. Providing the generation of a condensed intermediate poly-H-phosphonate oligonucleotide chain, which provides a phosphorus atom-modified nucleic acid (referred to as Route B in the literature, Scheme 6, p. 36). In addition, the H-phosphonate oligonucleotide chains of Wada II are shorter in length (eg, dimer, trimmer, or tetramer). Capping Steps, Path B does not include a capping step, which, combined with the fact that it generally exhibits low purity as a result of the accumulation of "n-1" type by-products, makes the Wada II pathway more Limitations are included with respect to the synthesis of long oligonucleotides. Wada II generally contemplates that certain oligonucleotides may contain different modifications at each bound phosphate, whereas Wada II repeatedly incorporates such controlled modifications. The methods described herein are not described or suggested. Wada II describes a synthetic cycle that does not require complete incorporation of the H-phosphonate intermediate oligonucleotide prior to modification with bound phosphate (in the literature, Route A, p. 35). , Scheme 5, "Synthesis of Nucleic Acid Containing the Chiral X-Phosphate moiety of Formula 1 Through Route A"), this general disclosure is necessary to incorporate certain P-modifications provided by the present invention. It does not teach the important steps to be taken, and this cycle is efficient and diverse enough to be useful in the synthesis of chiral-controlled P-modified oligonucleotides, especially longer oligonucleotides. I'm not doing it either.

At least one such inefficiency in Wada II is described by Wada et al International Publication No. 2012/039448 (“Wada III”). Wada III discloses that novel chiral auxiliary is used in the method of Wada II to generate H-phosphonate oligonucleotides once constructed and continuously modified to obtain, among other things, phosphorothioates and the like. Wada et al. Confirmed in Wada III that the four types of chiral auxiliary disclosed in Wada II cannot be efficiently removed because they form a strong bond with phosphorus in the bound phosphate. .. Wada III states that removing the chiral auxiliary of Wada II requires strict conditions that tend to impair the integrity of oligonucleotide products. Wada III has confirmed that this is a particular problem when synthesizing long-chain oligonucleotides. The reason is that, at least as the degradation reaction proceeds, it further reacts with the oligonucleotide product to produce additional by-products that degrade the oligonucleotide product. Therefore, Wada III uses the mechanism of S _N 1 to open the H-phosphonate oligonucleotide bond under weakly acidic conditions (Route B) or through the β-removal pathway under relatively weakly basic conditions. We provide a chiral auxiliary that can efficiently cleave from oligonucleotides.

Those skilled in the art of chemistry and synthesis will readily appreciate the complexity associated with the production of chiral-controlled oligonucleotides provided by the present invention, for example. For example, in order to synthesize and isolate a chirally controlled oligonucleotide, the conditions for each monomer addition should be designed to be as follows: (1) The chemistry is compatible with each portion of the increasing oligonucleotide; (2) The by-products produced during the addition of each monomer are the constitutive and stereochemical integration of the increasing oligonucleotide. And (3) the composition of the final crude product is a composition that isolates the desired chiral-controlled oligonucleotide product.

Oligonucleotide phosphorothioates have shown potential therapeutic potential (Stein et al., Science (1993), 261: 1004-12; Agrawal et al., Antisense Res. And Dev. (1992), 2: 261. -66; Bayever et al., Antisense Res. And Dev. (1993), 3: 383-390). Oligonucleotide phosphorothioates prepared independently of the stereochemistry of phosphorothioates exist as a mixture of ²ⁿ diastereomers, where n is the number of internucleotide phosphorothioate bonds. The chemical and biological properties of these diastereomeric phosphorothioates can be distinctly different from others. For example, Wada et al. (Nucleic Acids Symposium Series No. 51 p. 119-120; doi: 10.1093 / nas / nrm060) were sterically defined-(Rp)-(Ups) 9U / (Ap) 9A. The double strand exhibits a higher Tm value than the normal-(Up) 9U / (Ap) 9A and the sterically defined-(Sp)-(Ups) 9U that did not form a double strand. I found. In another example, the study by Tang et al. (Nucleosides Nucleosides (1995), 14: 985-990) found that sterically pure Rp oligodeoxyribonucleoside phosphorothioates were more than parental oligodeoxyribonucleoside phosphorothioates with undefined linkages. Was also found to have low stability to the nucleases inherent in human sera.

Chiral-controlled oligonucleotides and chiral-controlled oligonucleotide compositions The present invention provides chiral-controlled oligonucleotides of crude and high-purity and high-diastereomer purity, and chiral-controlled oligonucleotide compositions. In some embodiments, the present invention provides chirally controlled oligonucleotides with high crude purity and chirally controlled oligonucleotide compositions. In some embodiments, the present invention provides chiral-controlled oligonucleotides and high-purity oligonucleotide compositions of chiral-controlled diastereomers.

In some embodiments, the invention comprises at least one type of oligonucleotides, each of which is: 1) base sequence; 2) skeletal binding pattern; 3) skeletal chiral center pattern; and 4) A chiral-controlled composition defined by a skeletal P-modification pattern.

In some embodiments, the invention comprises multiple oligonucleotides of the same type, each of which is: 1) base sequence; 2) skeletal binding pattern; 3) skeletal chiral center pattern; and 4 ) A chiral controlled composition defined by a skeletal P-modification pattern. In some embodiments, the invention comprises two or more types of oligonucleotides, each of which is: 1) base sequence; 2) skeletal binding pattern; 3) skeletal chiral center pattern; and 4) A chiral-controlled composition defined by a skeletal P-modification pattern.

In some embodiments, the invention provides an oligonucleotide that comprises one or more diastereomerically pure internucleotide linkages with respect to chirally bound phosphates. In some embodiments, the invention provides an oligonucleotide containing one or more diastereomerically pure internucleotide bonds having the structure of formula I. In some embodiments, the invention provides oligonucleotides containing one or more diastereomerically pure internucleotide bonds and one or more phosphodiester bonds with respect to chiral-bound phosphates. In some embodiments, the present invention provides oligonucleotides comprising one or more diastereomerically pure structures of formula I, one or more, and one or more phosphate diester bonds. In some embodiments, the present invention provides oligonucleotides comprising one or more diastereomerically pure structures of formula I-c, one or more oligonucleotide bonds, and one or more phosphate diester bonds. In some embodiments, such oligonucleotides are prepared using stereoselective oligonucleotide synthesis and diastereomerically pre-designed for chirally bound phosphates, as described in this application. Form a pure oligonucleotide bond. For example, (Rp / Sp, Rp / Sp, Rp / Sp, Rp, Rp, Sp, Sp, Sp, Sp, SpSp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp) -d [GsCsCsTsCsAsGsTsCsCsCsCsCsCsCsTs In one exemplary oligonucleotide represented by, the first three oligonucleotide bonds are made using conventional oligonucleotide synthesis methods, and diastereomerically pure oligonucleotide bonds are described in this application. As you can see, it is produced by stereochemical control. Exemplary internucleotide bindings include those having the structure of Formula I, which are further described below. In some embodiments, such oligonucleotides further include, but are limited to, the sequences described in this application, including those described in Tables 2 and 4, and Annex A, B, and C. It's not a thing.

In some embodiments, the oligonucleotides provided include a combination of sterically pure and sterically random oligonucleotide linkages with respect to chirality at the bound phosphate. For example, in some embodiments, having one or more blocks of sterically defined internucleotide binding within an oligonucleotide that is otherwise sterically random with respect to chirality at bound phosphate. Is desirable. In some embodiments, it is desirable to have one or more blocks of sterically random internucleotide binding within the oligonucleotide, which is otherwise sterically defined with respect to chirality at the bound phosphate. ..

In some embodiments, at least one nucleotide unit of the provided oligonucleotide is incorporated using the stereoselective oligonucleotide synthesis described in this application and is pre-designed for chirally bound phosphates. It forms a diastereomerically pure oligonucleotide bond. In some embodiments, at least two nucleotide units of the provided oligonucleotides have been incorporated using stereoselective oligonucleotide synthesis described in this application and are pre-designed for chirally bound phosphates. It forms at least two diastereomerically pure oligonucleotide bonds. In some embodiments, at least three nucleotide units of the provided oligonucleotides have been incorporated using stereoselective oligonucleotide synthesis described in this application and are pre-designed for chirally bound phosphates. It forms at least three diastereomerically pure oligonucleotide bonds. In some embodiments, at least one, two, or three pre-designed diastereomerically pure nucleotide bonds are flanking each other. In some embodiments, at least one, two, or three pre-designed diastereomerically pure internucleotide bonds are not adjacent to each other.

In some embodiments, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the nucleotide units of the oligonucleotide provided are. Incorporated using the stereoselective oligonucleotide synthesis described in this application, it forms a pre-designed diastereomerically pure polynucleotide bond for chirally bound phosphates. As described herein, in some embodiments, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% nucleotide units. It occurs in one or more blocks and provides blockmers. In some embodiments, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% nucleotide units occur in alternating patterns. Providing altmers. Those skilled in the art of the art will appreciate that any desirable pattern is feasible using the methods of the invention discussed herein.

In some embodiments, the invention provides a chirally controlled oligonucleotide in which at least two of the individual internucleotide bonds within the oligonucleotide have different stereochemistry and / or different P-modifications from each other. In certain embodiments, the present invention provides chirally controlled oligonucleotides, in which at least two individual polynucleotide linkages within the oligonucleotide have different P-modifications from each other. In certain embodiments, the present invention provides a chiral-controlled oligonucleotide in which at least two of the individual internucleotide bonds within the oligonucleotide have different P-modifications with each other and are chiral-controlled oligonucleotides. The nucleotide contains at least one phosphate diester oligonucleotide bond. In certain embodiments, the invention provides a chiral-controlled oligonucleotide in which at least two of the individual internucleotide bonds within the oligonucleotide have different P-modifications with each other and are chiral-controlled oligos. Nucleotides include at least one phosphate diester polynucleotide bond and at least one phosphodiester internucleotide bond. In certain embodiments, the present invention provides chirally controlled oligonucleotides, in which at least two of the individual polynucleotide linkages within the oligonucleotide have different P-modifications with each other and are chirally controlled oligonucleotides. The nucleotide contains at least one phosphorothioate triester oligonucleotide binding. In certain embodiments, the present invention provides a chiral-controlled oligonucleotide in which at least two of the individual internucleotide bonds within the oligonucleotide have different P-modifications with each other and are chiral-controlled oligonucleotides. Nucleotides include at least one phosphate diester polynucleotide binding and at least one phosphorothioate triester oligonucleotide binding.

の構造を有する１以上の修飾インターヌクレオチド結合を含むキラル制御されたオリゴヌクレオチドを提供し、ここで、各変数は、下記のとおり定義され、記載するとおりである。いくつかの実施形態において、本発明は、式Ｉの１個以上の修飾されたインターヌクレオチド結合を含むキラル制御されたオリゴヌクレオチドを提供し、オリゴヌクレオチド内の式Ｉの個々のインターヌクレオチド結合が互いに異なるＰ－修飾を有する。いくつかの実施形態において、本発明は、式Ｉの１個以上の修飾されたインターヌクレオチド結合を含むキラル制御されたオリゴヌクレオチドを提供し、オリゴヌクレオチド内の式Ｉの個々のインターヌクレオチド結合は、互いに異なる－Ｘ－Ｌ－Ｒ^１を有する。いくつかの実施形態において、本発明は、式Ｉの１個以上の修飾されたインターヌクレオチド結合を含むキラル制御されたオリゴヌクレオチドを提供し、オリゴヌクレオチド内の式Ｉの個々のインターヌクレオチド結合は、互いに異なるＸを有する。いくつかの実施形態において、本発明は、式Ｉの１個以上の修飾されたインターヌクレオチド結合を含むキラル制御されたオリゴヌクレオチドを提供し、オリゴヌクレオチド内の式Ｉの個々のインターヌクレオチド結合は、互いに異なる－Ｌ－Ｒ^１を有する。 In certain embodiments, the present invention independently formulates I:

Provided are chirally controlled oligonucleotides comprising one or more modified oligonucleotide linkages having the structure of, wherein each variable is defined and described as follows. In some embodiments, the invention provides a chirally controlled oligonucleotide containing one or more modified oligonucleotide linkages of formula I, in which the individual internucleotide linkages of formula I within the oligonucleotide are attached to each other. Has different P-modifications. In some embodiments, the invention provides a chirally controlled oligonucleotide containing one or more modified oligonucleotide linkages of formula I, wherein the individual polynucleotide linkages of formula I within the oligonucleotide are. It has different -X-L-R ^1s . In some embodiments, the invention provides a chirally controlled oligonucleotide containing one or more modified oligonucleotide linkages of formula I, wherein the individual polynucleotide linkages of formula I within the oligonucleotide are. It has different Xs from each other. In some embodiments, the invention provides a chirally controlled oligonucleotide containing one or more modified oligonucleotide linkages of formula I, wherein the individual polynucleotide linkages of formula I within the oligonucleotide are. It has different —LR ^1s from each other.

In some embodiments, the invention provides a chirally controlled oligonucleotide in which at least two of the individual internucleotide bonds within the oligonucleotide have different stereochemistry and / or different P-modifications from each other. In some embodiments, the invention provides a chiral-controlled oligonucleotide in which at least two of the individual internucleotide bonds within the oligonucleotide have different stereochemistry from each other and are at least a chiral-controlled oligonucleotide. Part of the structure of the nucleotide is characterized by a repeating pattern of alternating stereochemistry.

In some embodiments, the invention provides a chiral-controlled oligonucleotide in which at least two of the individual internucleotide bonds within the oligonucleotide have different X atoms in ^one part of the -XLR, and / or. They have different P-modifications to each other in the sense that they have different L groups in ^one part of -XLR and / or different ^R1 atoms in ^one part of XLR.

In some embodiments, the invention provides a chirally controlled oligonucleotide in which at least two of the individual polynucleotide linkages within the oligonucleotide have different stereochemistry and / or different P-modifications from each other. , Oligonucleotides have the following formula:
[S ^B n1R ^B n2S ^B n3R ^B n4. .. .. SB ^{nxR B ny} ]
Has a structure represented by
Here, each RB independently represents a block of nucleotide units having an ^R configuration in the bound phosphate;
Each SB independently represents a block of nucleotide units with an S ^{configuration} in the bound phosphate;
Oligonucleotides are different from each other in steric chemistry by being zero or an integer, each of n1 to ny, with the requirement that at least one odd n and at least one even n are non-zero numbers. Includes at least two individual oligonucleotide bonds; and where the sum of n1-ny is 2-200, and in some embodiments, the lower bounds are 2, 3, 4, 5, 6 , 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25 Selected from a group of 25 or more, with an upper limit of 5 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, and 200 or more, with an upper limit greater than the lower limit.

In some such embodiments, each n has the same value; in some embodiments, each even n has the same value as each other n; in some embodiments, each odd n Are the same values as odd n with each other; in some embodiments, at least two even n are different values from each other; in some embodiments, at least two odd n are different values from each other. Is.

In some embodiments, at least two adjacent ns are equal to each other and the oligonucleotide provided comprises adjacent blocks of S stereochemical bonds and R stereochemical bonds of equal length. In some embodiments, the oligonucleotides provided comprise a repeating block of S and R stereochemical bonds of equal length. In some embodiments, the provided oligonucleotides contain repeating blocks of S and R stereochemical bonds, and at least two such blocks are of different lengths from each other; some such embodiments. In the form, each S stereochemical block has the same length and is different from the length of each R stereochemistry, and the length of each R stereochemistry may be arbitrarily the same length as each other.

In some embodiments, at least two n adjacent to each other, skipping other than n, are equal to each other, and the oligonucleotides provided contain at least two blocks of first stereochemical bonds of equal length to each other. It is divided into blocks of stereochemical bonds, and the divided blocks may have the same length as or different from the blocks of the first stereochemistry.

In some embodiments, n associated with the binding block at the end of the provided oligonucleotide is of the same length. In some embodiments, the oligonucleotides provided have the same binding stereochemical terminal block. In some such embodiments, the terminal blocks are separated from each other by another intermediate block of bound stereochemistry.

In some embodiments, the formula [S ^B n1R ^B n2S ^B n3R ^B n4. .. .. The oligonucleotide shown and provided by SB ^{nxR B ny} ] is a stereoblockmer. In some embodiments, the formula [S ^B n1R ^B n2S ^B n3R ^B n4. .. .. SB ^{nxR B ny} ], the oligonucleotide provided is a stereo skipmer. In some embodiments, the formula [S ^B n1R ^B n2S ^B n3R ^B n4. .. .. The oligonucleotides shown and provided by SB ^{nxR B ny} ] are stereoaltomers. In some embodiments, the formula [S ^B n1R ^B n2S ^B n3R ^B n4. .. .. The oligonucleotide shown and provided by SB ^{nxR B ny} ] is a gapmer.

In some embodiments, the formula [S ^B n1R ^B n2S ^B n3R ^B n4. .. .. The oligonucleotides shown and provided in SB ^{nxR B ny} ] may be any of the patterns described above and further include a pattern of P-modification. For example, in some embodiments, the formula [S ^B n1R ^B n2S ^B n3R ^B n4. .. .. SB nxR ^B ny], the oligonucleotides provided are stereo skipmers and ^P -modified skipmers. In some embodiments, the formula [S ^B n1R ^B n2S ^B n3R ^B n4. .. .. The oligonucleotides shown and provided by SB nxR ^B ny] are stereoblockmers and ^P -modified altomers. In some embodiments, the formula [S ^B n1R ^B n2S ^B n3R ^B n4. .. .. The oligonucleotides shown and provided by SB nxR ^B ny] are stereoaltomers and ^P -modified blockmers.

の構造を有する１以上の修飾されたインターヌクレオチドの結合を含むキラル制御されたオリゴヌクレオチドであり、
ここで、Ｐ^＊は、不斉リン原子であり、ＲｐまたはＳｐのいずれかであり；
Ｗは、Ｏ、ＳまたはＳｅであり；
Ｘ、ＹおよびＺのそれぞれは、独立して－Ｏ－、－Ｓ－、－Ｎ（－Ｌ－Ｒ^１）－、またはＬであり；
Ｌは、共有結合または置換されていてもよい、直鎖または分岐のＣ_１～Ｃ_１０アルキレンであり、ここでＬの１以上のメチレン単位は、置換されていてもよいＣ_１～Ｃ_６アルキレン、Ｃ_１～Ｃ_６アルケニレン、－Ｃ≡Ｃ－、－Ｃ（Ｒ´）_２－、－Ｃｙ－、－Ｏ－、－Ｓ－、－Ｓ－Ｓ－、－Ｎ（Ｒ´）－、－Ｃ（Ｏ）－、－Ｃ（Ｓ）－、－Ｃ（ＮＲ´）－、－Ｃ（Ｏ）Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｃ（Ｏ）Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｃ（Ｏ）－、－Ｎ（Ｒ´）Ｃ（Ｏ）Ｏ－、－ＯＣ（Ｏ）Ｎ（Ｒ´）－、－Ｓ（Ｏ）－、－Ｓ（Ｏ）_２－、－Ｓ（Ｏ）_２Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｓ（Ｏ）_２－、－ＳＣ（Ｏ）－、－Ｃ（Ｏ）Ｓ－、－ＯＣ（Ｏ）－、または－Ｃ（Ｏ）Ｏ－により任意におよび独立して置換され；
Ｒ^１は、ハロゲン、Ｒ、または置換されていてもよいＣ_１～Ｃ_５０脂肪族であり、１以上のメチレン単位は、置換されていてもよいＣ_１～Ｃ_６アルキレン、Ｃ_１～Ｃ_６アルケニレン、－Ｃ≡Ｃ－、－Ｃ（Ｒ´）_２－、－Ｃｙ－、－Ｏ－、－Ｓ－、－Ｓ－Ｓ－、－Ｎ（Ｒ´）－、－Ｃ（Ｏ）－、－Ｃ（Ｓ）－、－Ｃ（ＮＲ´）－、－Ｃ（Ｏ）Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｃ（Ｏ）Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｃ（Ｏ）－、－Ｎ（Ｒ´）Ｃ（Ｏ）Ｏ－、－ＯＣ（Ｏ）Ｎ（Ｒ´）－、－Ｓ（Ｏ）－、－Ｓ（Ｏ）_２－、－Ｓ（Ｏ）_２Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｓ（Ｏ）_２－、－ＳＣ（Ｏ）－、－Ｃ（Ｏ）Ｓ－、－ＯＣ（Ｏ）－、または－Ｃ（Ｏ）Ｏ－により任意におよび独立して置換され；
各Ｒ´は、独立して－Ｒ、－Ｃ（Ｏ）Ｒ、－ＣＯ_２Ｒ、もしくは－ＳＯ_２Ｒであるか、または：
同じ窒素上の２つのＲ´は、それらの介在原子と一緒になって置換されていてもよいヘテロ環式、もしくはヘテロアリール環を形成するか、または
同じ炭素上の２つのＲ´は、それらの介在原子と一緒になって、置換されていてもよいアリール、炭素環式、ヘテロ環式、またはヘテロアリール環を形成し；
－Ｃｙ－は、フェニレン、カルボシクリレン、アリーレン、ヘテロアリーレン、またはヘテロシクリレンから選択される置換されていてもよい２価の環であり；
各Ｒは、独立して水素である、または、Ｃ_１～Ｃ_６脂肪族、フェニル、カルボシクリル、アリール、ヘテロアリール、またはヘテロシクリルから選択される置換されていてもよい基であり；および各

は、独立してヌクレオシドとの結合を表す。 In some embodiments, the formula [S ^B n1R ^B n2S ^B n3R ^B n4. .. .. The oligonucleotides provided, represented by SB ^{nxR B ny} ], are independently of formula I :.

A chirally controlled oligonucleotide containing the binding of one or more modified oligonucleotides having the structure of.
Where P ^* is an asymmetric phosphorus atom, either Rp or Sp;
W is O, S or Se;
Each of X, Y and Z is independently -O-, -S-, -N (-L-R ¹ )-, or L;
L is a linear or branched C ₁ to C ₁₀ alkylene that may be covalently bonded or substituted, wherein one or more methylene units of L may be substituted C ₁ to C ₆ alkylene. , C ₁ to C ₆ alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R')-,- C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-, -S (O) ₂ -, -S (O) ₂ N (R')-,-N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O)-, Or optionally and independently replaced by -C (O) O-;
R ¹ is a halogen, R, or optionally substituted C ₁ to C ₅₀ aliphatic, and one or more methylene units may be substituted C ₁ to C ₆ alkylene, C ₁ to C ₆ Alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R')-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-, -S (O) _2- , -S (O) ) ₂ N (R')-,-N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O)-, or -C (O) Replaced arbitrarily and independently by O-;
Each R'is independently -R, -C (O) R, -CO ₂ R, or -SO ₂ R, or:
Two R'on the same nitrogen form a heterocyclic or heteroaryl ring that may be substituted together with their intervening atoms, or two R'on the same carbon are them. Together with the intervening atoms of, it forms an aryl, carbocyclic, heterocyclic, or heteroaryl ring that may be substituted;
-Cy- is a optionally substituted divalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene;
Each R is an independently hydrogen or is _a optionally substituted group selected from _C1-6 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl; and each

Represents the binding to the nucleoside independently.

In some embodiments, the chirally controlled oligonucleotide comprises a phosphate bond of one or more modified polynucleotides. In some embodiments, the chirally controlled oligonucleotide comprises, for example, a phosphorothioate or a phosphorothioate triester bond. In some embodiments, the chirally controlled oligonucleotide comprises a phosphorothioate triester bond. In some embodiments, the chirally controlled oligonucleotide comprises at least two phosphorothioate triester bonds. In some embodiments, the chirally controlled oligonucleotide contains at least three phosphorothioate triester bonds. In some embodiments, the chirally controlled oligonucleotide comprises at least 4 phosphorothioate triester bonds. In some embodiments, the chirally controlled oligonucleotide comprises at least 5 phosphorothioate triester bonds. Phosphate bonds of such exemplary modified polynucleotides are further described herein.

In some embodiments, the chirally controlled oligonucleotide contains phosphate bonds of different polynucleotides. In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphate diester polynucleotide bond and at least one modified polynucleotide bond. In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphate diester polynucleotide bond and at least one phosphodiester bond. In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphate diester polynucleotide bond and at least two phosphodiester triester bonds. In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphate diester internucleotide bond and at least three phosphodiester triester bonds. In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphate diester internucleotide bond and at least four phosphodiester triester bonds. In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphate diester polynucleotide bond and at least 5 phosphodiester triester bonds. Phosphate bonds of such exemplary modified polynucleotides are further described herein.

In some embodiments, the phosphorothioate triester bond comprises, for example, a chiral auxiliary used to control the stereoselectivity of the reaction. In some embodiments, the phosphorothioate triester bond is free of chiral auxiliary. In some embodiments, the phosphorothioate triester bond is intentionally sustained until and / or throughout administration to the subject.

In some embodiments, the chirally controlled oligonucleotide is attached to a solid support. In some embodiments, the chirally controlled oligonucleotide is cleaved from the solid support.

In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphate diester polynucleotide bond and at least two consecutively modified polynucleotide bonds. In some embodiments, the chirally controlled oligonucleotide comprises at least one phosphate diester polynucleotide bond and at least two contiguous phosphorothioate triester oligonucleotide bonds.

In some embodiments, the chirally controlled oligonucleotide is blockmer. In some embodiments, the chirally controlled oligonucleotide is a stereoblockmer. In some embodiments, the chirally controlled oligonucleotide is a P-modified blocker. In some embodiments, the chirally controlled oligonucleotide is a binding blocker.

In some embodiments, the chirally controlled oligonucleotide is an altomer. In some embodiments, the chirally controlled oligonucleotide is a stereoaltomer. In some embodiments, the chirally controlled oligonucleotide is a P-modified altomer. In some embodiments, the chirally controlled oligonucleotide is a bound altomer.

In some embodiments, the chirally controlled oligonucleotide is a unimmer. In some embodiments, the chirally controlled oligonucleotide is a stereo-unimer. In some embodiments, the chirally controlled oligonucleotide is a P-modified unimer. In some embodiments, the chirally controlled oligonucleotide is a binding unimmer.

In some embodiments, the chirally controlled oligonucleotide is a gapmer.

In some embodiments, the chirally controlled oligonucleotide is Skipmer.

の構造を独立して有する１以上の修飾されたインターヌクレオチドの結合を含むキラル制御されたオリゴヌクレオチドを提供し、
ここで、Ｐ^＊は、不斉リン原子であり、ＲｐまたはＳｐのいずれかであり；
Ｗは、Ｏ、ＳまたはＳｅであり；
Ｘ、ＹおよびＺのそれぞれは、独立して－Ｏ－、－Ｓ－、－Ｎ（－Ｌ－Ｒ^１）－、またはＬであり；
Ｌは、共有結合または置換されていてもよい、直鎖または分岐のＣ_１～Ｃ_１０アルキレンであり、ここでＬの１以上のメチレン単位は、置換されていてもよいＣ_１～Ｃ_６アルキレン、Ｃ_１～Ｃ_６アルケニレン、－Ｃ≡Ｃ－、－Ｃ（Ｒ´）_２－、－Ｃｙ－、－Ｏ－、－Ｓ－、－Ｓ－Ｓ－、－Ｎ（Ｒ´）－、－Ｃ（Ｏ）－、－Ｃ（Ｓ）－、－Ｃ（ＮＲ´）－、－Ｃ（Ｏ）Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｃ（Ｏ）Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｃ（Ｏ）－、－Ｎ（Ｒ´）Ｃ（Ｏ）Ｏ－、－ＯＣ（Ｏ）Ｎ（Ｒ´）－、－Ｓ（Ｏ）－、－Ｓ（Ｏ）_２－、－Ｓ（Ｏ）_２Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｓ（Ｏ）_２－、－ＳＣ（Ｏ）－、－Ｃ（Ｏ）Ｓ－、－ＯＣ（Ｏ）－、または－Ｃ（Ｏ）Ｏ－により任意におよび独立して置換され；
Ｒ^１は、ハロゲン、Ｒ、または置換されていてもよいＣ_１～Ｃ_５０脂肪族であり、１以上のメチレン単位は、置換されていてもよいＣ_１～Ｃ_６アルキレン、Ｃ_１～Ｃ_６アルケニレン、－Ｃ≡Ｃ－、－Ｃ（Ｒ´）_２－、－Ｃｙ－、－Ｏ－、－Ｓ－、－Ｓ－Ｓ－、－Ｎ（Ｒ´）－、－Ｃ（Ｏ）－、－Ｃ（Ｓ）－、－Ｃ（ＮＲ´）－、－Ｃ（Ｏ）Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｃ（Ｏ）Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｃ（Ｏ）－、－Ｎ（Ｒ´）Ｃ（Ｏ）Ｏ－、－ＯＣ（Ｏ）Ｎ（Ｒ´）－、－Ｓ（Ｏ）－、－Ｓ（Ｏ）_２－、－Ｓ（Ｏ）_２Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｓ（Ｏ）_２－、－ＳＣ（Ｏ）－、－Ｃ（Ｏ）Ｓ－、－ＯＣ（Ｏ）－、または－Ｃ（Ｏ）Ｏ－により任意におよび独立して置換され；
各Ｒ´は、独立して－Ｒ、－Ｃ（Ｏ）Ｒ、－ＣＯ_２Ｒ、もしくは－ＳＯ_２Ｒであるか、または：
同じ窒素上の２つのＲ´は、それらの介在原子と一緒になって置換されていてもよいヘテロ環式、もしくはヘテロアリール環を形成するか、または
同じ炭素上の２つのＲ´は、それらの介在原子と一緒になって、置換されていてもよいアリール、炭素環式、ヘテロ環式、またはヘテロアリール環を形成し；
－Ｃｙ－は、フェニレン、カルボシクリレン、アリーレン、ヘテロアリーレン、またはヘテロシクリレンから選択される置換されていてもよい２価の環であり；
各Ｒは、独立して水素である、または、Ｃ_１～Ｃ_６脂肪族、フェニル、カルボシクリル、アリール、ヘテロアリール、またはヘテロシクリルから選択される置換されていてもよい基であり；および各

は、独立してヌクレオシドとの結合を表す。 In some embodiments, the present invention relates to Formula I :.

Provided are chirally controlled oligonucleotides containing the binding of one or more modified oligonucleotides that independently have the structure of.
Where P ^* is an asymmetric phosphorus atom, either Rp or Sp;
W is O, S or Se;
Each of X, Y and Z is independently -O-, -S-, -N (-L-R ¹ )-, or L;
L is a linear or branched C ₁ to C ₁₀ alkylene that may be covalently bonded or substituted, wherein one or more methylene units of L may be substituted C ₁ to C ₆ alkylene. , C ₁ to C ₆ alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R')-,- C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-, -S (O) ₂ -, -S (O) ₂ N (R')-,-N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O)-, Or optionally and independently replaced by -C (O) O-;
R ¹ is a halogen, R, or optionally substituted C ₁ to C ₅₀ aliphatic, and one or more methylene units may be substituted C ₁ to C ₆ alkylene, C ₁ to C ₆ Alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R')-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-, -S (O) _2- , -S (O) ) ₂ N (R')-,-N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O)-, or -C (O) Replaced arbitrarily and independently by O-;
Each R'is independently -R, -C (O) R, -CO ₂ R, or -SO ₂ R, or:
Two R'on the same nitrogen form a heterocyclic or heteroaryl ring that may be substituted together with their intervening atoms, or two R'on the same carbon are them. Together with the intervening atoms of, it forms an aryl, carbocyclic, heterocyclic, or heteroaryl ring that may be substituted;
-Cy- is a optionally substituted divalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene;
Each R is an independently hydrogen or is _a optionally substituted group selected from _C1-6 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl; and each

Represents the binding to the nucleoside independently.

Generally, as described above and herein, P ^* is an asymmetric phosphorus atom, either Rp or Sp. In some embodiments, P ^* is Rp. In another embodiment, P ^* is Sp. In some embodiments, the oligonucleotide comprises one or more oligonucleotide bonds of formula I, where each P ^* is independently Rp or Sp. In some embodiments, the oligonucleotide contains one or more oligonucleotide bonds of formula I, where each P ^* is Rp. In some embodiments, the oligonucleotide contains one or more oligonucleotide bonds of formula I, where each P ^* is Sp. In some embodiments, the oligonucleotide contains at least one polynucleotide bond of formula I, where P ^* is Rp. In some embodiments, the oligonucleotide contains at least one polynucleotide bond of formula I, where P ^* is Sp. In some embodiments, the oligonucleotide comprises at least one polynucleotide binding of formula I, where P ^* is Rp, and P ^* comprises at least one polynucleotide binding of formula I, which is Sp.

Generally, as described above and herein, W is O, S, or Se. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, W is Se. In some embodiments, the oligonucleotide contains at least one polynucleotide bond of formula I, where W is O. In some embodiments, the oligonucleotide comprises at least one polynucleotide bond of formula I, where W is S. In some embodiments, the oligonucleotide contains at least one polynucleotide bond of formula I, where W is Se.

Generally, as described above and herein, each R is independently substituted with hydrogen or selected from C ₁ to C ₆ aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl. Is also a good base.

In some embodiments, R is hydrogen. _In some embodiments, R is _a optionally substituted group selected from C1-6 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl.

In some embodiments, R is an optionally substituted C ₁ to C ₆ aliphatic. In some embodiments, R is a _{optionally substituted C 1-6 alkyl} . In some embodiments, R is a linear or branched hexyl that may be substituted. In some embodiments, R is a linear or branched pentyl that may be substituted. In some embodiments, R is a linear or branched butyl that may be substituted. In some embodiments, R is linear or branched propyl, which may be substituted. In some embodiments, R is an ethyl that may be substituted. In some embodiments, R is a methyl that may be substituted.

In some embodiments, R is a optionally substituted phenyl. In some embodiments, R is a substituted phenyl. In some embodiments, R is phenyl.

In some embodiments, R is a carbocyclyl that may be substituted. In some embodiments, R is C ₃ to C ₁₀ carbocyclyl which may be substituted. In some embodiments, R is a monocyclic carbocyclyl that may be substituted. In some embodiments, R is a optionally substituted cycloheptyl. In some embodiments, R is a cyclohexyl that may be substituted. In some embodiments, R is cyclopentyl which may be substituted. In some embodiments, R is a optionally substituted cyclobutyl. In some embodiments, R is cyclopropyl which may be substituted. In some embodiments, R is a bicyclic carbocyclyl that may be substituted.

In some embodiments, R is an optionally substituted aryl. In some embodiments, R is a bicyclic aryl ring that may be substituted.

In some embodiments, R is a heteroaryl which may be substituted. In some embodiments, R is an optionally substituted 5- to 6-membered monocyclic heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, sulfur, or oxygen. be. In some embodiments, R is a substituted 5- to 6-membered monocyclic heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an unsubstituted 5- to 6-membered monocyclic heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, sulfur, or oxygen.

In some embodiments, R is a optionally substituted 5-membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen or sulfur. In some embodiments, R is an optionally substituted 6-membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R is a optionally substituted 5-membered monocyclic heteroaryl ring having one heteroatom selected from nitrogen, oxygen or sulfur. In some embodiments, R is selected from pyrrolyl, furanyl, or thienyl.

In some embodiments, R is a optionally substituted 5-membered heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R is a optionally substituted 5-membered heteroaryl ring having one nitrogen atom, the additional heteroatom being selected from sulfur or oxygen. Exemplary R groups include optionally substituted pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl or isooxazolyl.

In some embodiments, R is a 6-membered heteroaryl ring with 1-3 nitrogen atoms. In another embodiment, R is a optionally substituted 6-membered heteroaryl ring having 1-2 nitrogen atoms. In some embodiments, R is a optionally substituted 6-membered heteroaryl ring having two nitrogen atoms. In certain embodiments, R is a optionally substituted 6-membered heteroaryl ring having one nitrogen. Exemplary R groups are pyridinyl, pyrimidinyl, pyrazinyl, pyridadinyl, triazinyl, or tetrazinyl which may be substituted.

In certain embodiments, R is an optionally substituted 8- to 10-membered bicyclic heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. .. In some embodiments, R is a optionally substituted 5,6-condensed heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In another embodiment, R is a optionally substituted 5,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R is a optionally substituted 5,6-condensed heteroaryl ring having one heteroatom independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an indrill that may be substituted. In some embodiments, R is an optionally substituted azabicyclo [3.2.1] octanyl. In certain embodiments, R is a optionally substituted 5,6-condensed heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an azaindolyl that may be substituted. In some embodiments, R is a optionally substituted benzimidazolyl. In some embodiments, R is a optionally substituted benzothiazolyl. In some embodiments, R is a optionally substituted benzoxazolyl. In some embodiments, R is an indazolyl that may be substituted. In certain embodiments, R is a optionally substituted 5,6-condensed heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, R is a optionally substituted 6,6-condensed heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is a optionally substituted 6,6-condensed heteroaryl ring having one or two heteroatoms independently selected from nitrogen, oxygen, or sulfur. In another embodiment, R is a optionally substituted 6,6-condensed heteroaryl ring having one heteroatom independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is a quinolinyl that may be substituted. In some embodiments, R is an isoquinolinyl that may be substituted. From one aspect, R is a optionally substituted 6,6-condensed heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is quinazoline or quinoxaline.

In some embodiments, R is a heterocyclyl that may be substituted. In some embodiments, R is 3- to 7-membered saturated or partially unsaturated, which may be substituted with 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. It is a heterocyclic ring. In some embodiments, R is a substituted 3- to 7-membered saturated or partially unsaturated heterocyclic compound with 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. It is a ring. In some embodiments, R is an unsaturated 3- to 7-membered saturated or partially unsaturated heterocyclic compound having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. It is a ring.

In some embodiments, R is a heterocyclyl that may be substituted. In some embodiments, R is a 6-membered saturated or partially unsaturated heterocycle that may be substituted with 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. It is a formula ring. In some embodiments, R is a partially unsaturated 6-membered heterocyclic ring that may be substituted with two heteroatoms independently selected from nitrogen, oxygen, or sulfur. .. In some embodiments, R is a partially unsaturated 6-membered heterocyclic ring that may be substituted with two oxygen atoms.

In certain embodiments, R is a 3- to 7-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. .. In certain embodiments, R is oxylanyl, oxetanyl, tetrahydropyranyl, tetrahydropyranyl, oxepanyl, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepanyl, thiylanyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, thiepanyl, dioxolanyl, Oxazolidine, oxazolidinyl, imidazolidinyl, thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl, piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl, oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl, tetrahydropyranonyl, tetrahydropyran. Phenonyl, tetrahydrothiopyranonyl, thiepanonyl, oxazolidinonyl, oxadinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl, dioxepanonyl, oxathiolinonyl, oxathianonyl, oxathiepannyl, thiazolidinonyl, thiazinanoyl, thiazepanonyl, imidazolidinyl. , Diazepanonyl, imidazolidinedionyl, oxazolidinezionyl, thiazolidinezonyl, dioxolandionyl, oxathiolandionyl, piperazinzionyl, morpholindionyl, thiomorpholindionyl, tetrahydropyranyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, Piperidinyl, piperazinyl, pyrrolidinyl, tetrahydrothiophenyl, or tetrahydrothiopyranyl. In some embodiments, R is a 5-membered saturated or partially unsaturated heterocycle that may be substituted with 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. It is a formula ring.

In certain embodiments, R is a 5- to 6-member partially unsaturated monocycle that may be substituted with 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. It is a formula ring. In certain embodiments, R is a optionally substituted tetrahydropyridinyl, dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

In some embodiments, R may be substituted with 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 8- to 10-membered bicyclic saturated or partially saturated. It is an unsaturated heterocyclic ring. In some embodiments, R is an indolinyl that may be substituted. In some embodiments, R is an isoindrinyl that may be substituted. In some embodiments, R is 1,2,3,4-tetrahydroquinoline which may be substituted. In some embodiments, R is 1,2,3,4-tetrahydroisoquinoline which may be substituted.

Generally, as described above and herein, each R'is independently -R, -C (O) R, -CO ₂ R, or -SO ₂ R, or:
Two R'on the same nitrogen form a heterocyclic or heteroaryl ring that may be substituted together with their intervening atoms, or two R'on the same carbon are them. Together with the intervening atoms of, it forms an aryl, carbocyclic, heterocyclic, or heteroaryl ring that may be substituted;

In some embodiments, R'is -R, -C (O) R, -CO ₂ R, or -SO ₂ R, where R is defined above and is as described herein. ..

In some embodiments, R'is -R, where R is as defined and described above and herein. Is. In some embodiments, R'is hydrogen.

In some embodiments, R'is -C (O) R, where R is defined above and is as described herein. In some embodiments, R'is −CO ₂ R, where R is defined above and is as described herein. In some embodiments, R'is −SO ₂ R, where R is defined above and is as described herein.

In some embodiments, two R'on the same nitrogen form a heterocyclic or heteroaryl ring that may be substituted together with their intervening atoms. In some embodiments, two R'on the same carbon together with their intervening atoms form an aryl, carbocyclic, heterocyclic, or heteroaryl ring that may be substituted. ..

Generally, as described above and herein, -Cy- is a optionally substituted divalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene.

In some embodiments, -Cy- is a optionally substituted phenylene. In some embodiments, -Cy- is a carbocyclylene that may be substituted. In some embodiments, -Cy- is an arylene which may be substituted. In some embodiments, -Cy- is a heteroarylene that may be substituted. In some embodiments, -Cy- is a heterocyclylene that may be substituted.

Generally, as described above and herein, each of X, Y and Z is independently -O-, -S-, -N (-L-R ¹ )-, or L, L and Each of ^R1 is independently defined as described above and is as described below.

In some embodiments, X is —O—. In some embodiments, X is —S—. In some embodiments, X is —O— or —S—. In some embodiments, the oligonucleotide comprises at least one conjugate of an polynucleotide of formula I, where X is —O—. In some embodiments, the oligonucleotide comprises at least one conjugate of an polynucleotide of formula I, where X is —S—. In some embodiments, the oligonucleotide comprises at least one polynucleotide bond of formula I, where X is —O— and contains at least one oligonucleotide of formula I, where X is. , -S-. In some embodiments, the oligonucleotide comprises at least one polynucleotide binding of formula I, where X is —O— and at least one of the formula I internucleotide bindings, where X is. —S—, containing at least one polynucleotide bond of formula _I , where L is a linear or branched C1 to _C10 alkylene, optionally substituted, and one or more methylenes of L. The units may be substituted C ₁ to C ₆ alkylene, C ₁ to C ₆ alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R')-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')-,-N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R') -, -S (O)-,-S (O) ₂ -,-S (O) ₂ N (R')-,-N (R') S (O) _2- , -SC (O)-, It is optionally and independently substituted by -C (O) S-, -OC (O)-, or -C (O) O-.

In some embodiments, X is -N (-L-R ¹ )-. In some embodiments, X is -N (R ¹ )-. In some embodiments, X is -N (R')-. In some embodiments, X is -N (R)-. In some embodiments, X is -NH-.

In some embodiments, X is L. In some embodiments, X is a covalent bond. In some embodiments, X is a linear or branched C ₁ to C ₁₀ alkylene that may be substituted, and one or more methylene units of L may be substituted C ₁ to C. ₆ alkylene, C ₁ to C ₆ alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -SS-, -N (R')- , -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R') -, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-,-S (O) ) _2- , -S (O) ₂ N (R')-, -N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O) -Or -C (O) O-is arbitrarily and independently substituted. In some embodiments, X is C ₁ to C ₁₀ alkylene or C ₁ to C ₁₀ alkenylene which may be substituted. In some embodiments, X is methylene.

In some embodiments, Y is —O—. In some embodiments, Y is —S—.

In some embodiments, Y is -N (-L-R ¹ )-. In some embodiments, Y is -N (R ¹ )-. In some embodiments, Y is -N (R')-. In some embodiments, Y is -N (R)-. In some embodiments, Y is -NH-.

In some embodiments, Y is L. In some embodiments, Y is a covalent bond. In some embodiments, Y may be substituted or linear or branched C ₁ to C ₁₀ alkylene, and one or more methylene units of L may be substituted C ₁ to. C ₆ alkylene, C ₁ to C ₆ alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R') -, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R' )-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-,-S ( O) _2- , -S (O) ₂ N (R')-, -N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O) )-Or-C (O) O-arbitrarily and independently. In some embodiments, Y is C ₁ to C ₁₀ alkylene or C ₁ to C ₁₀ alkenylene which may be substituted. In some embodiments, Y is methylene.

In some embodiments, Z is —O—. In some embodiments, Z is —S—.

In some embodiments, Z is -N (-L-R ¹ )-. In some embodiments, Z is -N (R ¹ )-. In some embodiments, Z is -N (R')-. In some embodiments, Z is -N (R)-. In some embodiments, Z is -NH-.

In some embodiments, Z is L. In some embodiments, Z is a covalent bond. In some embodiments, Z is a linear or branched C ₁ -C ₁₀ alkylene, which may be substituted, and one or more methylene units of L may be substituted C _1- . C ₆ alkylene, C ₁ to C ₆ alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R') -, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R' )-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-,-S ( O) _2- , -S (O) ₂ N (R')-, -N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O) )-Or-C (O) O-arbitrarily and independently. _In some embodiments, Z is C1 to _C10 alkylene or C1 to _{C10 alkenylene} which may be substituted. In some embodiments, Z is methylene.

Generally, as described above and herein, L is a covalently bonded or optionally substituted, linear or branched C ₁ to C ₁₀ alkylene, and one or more methylene units of L are substituted. May be good C ₁ to C ₆ alkylene, C ₁ to C ₆ alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-,- N (R')-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) ) N (R')-,-N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-,-S (O) -, -S (O) _2- , -S (O) ₂ N (R')-, -N (R') S (O) _2- , -SC (O)-, -C (O) S- , -OC (O)-, or -C (O) O-, arbitrarily and independently.

In some embodiments, L is a covalent bond. In some embodiments, L is optionally substituted, linear or branched C ₁ to C ₁₀ alkylene, and one or more methylene units of L may be substituted C ₁ to C ₆ alkylene. , C ₁ to C ₆ alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R')-,- C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-, -S (O) ₂ -, -S (O) ₂ N (R')-,-N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O)-, Alternatively, it is arbitrarily and independently replaced by -C (O) O-.

Ｃ_１～Ｃ_６アルキレン、Ｃ_１～Ｃ_６アルケニレン、カルボシクリレン、アリーレン、Ｃ_１～Ｃ_６ヘテロアルキレン、ヘテロシクリレン、およびヘテロアリーレンから選択され；
Ｖは、－Ｏ－、－Ｓ－、－ＮＲ’－、Ｃ（Ｒ’）_２、－Ｓ－Ｓ－、－Ｂ－Ｓ－Ｓ－Ｃ－、

、または、Ｃ_１～Ｃ_６アルキレン、アリーレン、Ｃ_１～Ｃ_６ヘテロアルキレン、ヘテロシクリレン、およびヘテロアリーレンから選択される置換されていてもよい基から選択され；
Ａは、＝Ｏ、＝Ｓ、＝ＮＲ’、または＝Ｃ（Ｒ’）_２であり；
ＢおよびＣのそれぞれは、独立して－Ｏ－、－Ｓ－、－ＮＲ’－、－Ｃ（Ｒ’）_２－、または、Ｃ_１～Ｃ_６アルキレン、カルボシクリレン、アリーレン、ヘテロシクリレン、またはヘテロアリーレンから選択される置換されていてもよい基であり；および
各Ｒ’は独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L has a structure of -L ¹ -V-, where L ¹ is a optionally substituted group:

Selected from C ₁ to C ₆ alkylene, C ₁ to C ₆ alkenylene, carbocyclylene, arylene, C ₁ to C ₆ heteroalkylene, heterocyclylene, and heteroarylene;
V is -O-, -S-, -NR'-, C (R') ₂ , -S-S-, -B-S-SC-,

, Or selected from optionally substituted groups selected from C ₁ to C ₆ alkylene, arylene, C ₁ to C ₆ heteroalkylene, heterocyclylene, and heteroarylene;
A is = O, = S, = NR', or = C (R') ₂ ;
Each of B and C is independently -O-, -S-, -NR'-, -C (R') _2- , or C ₁ to C ₆ alkylene, carbocyclylene, arylene, heterocyclylene. , Or a optionally substituted group selected from heteroarylene; and each R'is independently defined as described above and as described herein.

である。 In some embodiments, L ¹ is

Is.

であり、
ここで環Ｃｙ’は、置換されていてもよいアリーレン、カルボシクリレン、ヘテロアリーレン、またはヘテロシクリレンである。いくつかの実施形態において、Ｌ^１は、置換されていてもよい

である。いくつかの実施形態において、Ｌ^１は、

である。 In some embodiments, L ¹ is

And
Here, the ring Cy'is an arylene, a carbocyclylene, a heteroarylene, or a heterocyclylene which may be substituted. ^In some embodiments, L1 may be substituted.

Is. In some embodiments, L ¹ is

Is.

から選択され、硫黄原子はＶに結合されている。いくつかの実施形態において、Ｌ^１は、置換されていてもよい基

から選択され、炭素原子はＸに結合されている。 In some embodiments, L ¹ is associated with X. Is. In some embodiments, L ¹ is a optionally substituted group.

Selected from, the sulfur atom is attached to V. In some embodiments, L ¹ is a optionally substituted group.

Selected from, the carbon atom is bonded to X.

の構造を有し、
ここで、
Ｅは、－Ｏ－、－Ｓ－、－ＮＲ’－又は－Ｃ（Ｒ’）_２－であり；
- - -は単結合又は二重結合であり；
２つのＲ^Ｌ１は、これらが結合している２個の炭素原子と一緒になって、置換されていてもよいアリール、炭素環式、ヘテロアリール又はヘテロ環式環を形成し；各Ｒ’は独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

Has the structure of
here,
E is -O-, -S-, -NR'-or -C (R') _2- ;
---- is a single bond or a double bond;
The two ^RL1s together with the two carbon atoms to which they are attached form an optionally substituted aryl, carbocyclic, heteroaryl or heterocyclic ring; each R'is Independently defined above and as described herein.

の構造を有し、
ここで、
Ｇは、－Ｏ－、－Ｓ－、または－ＮＲ’であり；
- - -は単結合又は二重結合であり；および
２つのＲ^Ｌ１は、これらが結合している２個の炭素原子と一緒になって、置換されていてもよいアリール、Ｃ_３～Ｃ_１０炭素環式、ヘテロアリール又はヘテロ環式環を形成し。 In some embodiments, L is

Has the structure of
here,
G is -O-, -S-, or -NR';
---- are single or double bonds; and the two ^RL1s may be substituted with the two carbon atoms to which they are attached, C ₃ to C ₁₀ ; Form a carbocyclic, heteroaryl or heterocyclic ring.

の構造を有し、
ここでＥは、－Ｏ－、－Ｓ－、－ＮＲ’－又は－Ｃ（Ｒ’）_２－であり；
Ｄは、＝Ｎ－、＝Ｃ（Ｆ）－、＝Ｃ（Ｃｌ）－、＝Ｃ（Ｂｒ）－、＝Ｃ（Ｉ）－、＝Ｃ（ＣＮ）－、＝Ｃ（ＮＯ_２）－、＝Ｃ（ＣＯ_２－（Ｃ_１～Ｃ_６脂肪族））－、または＝Ｃ（ＣＦ_３）－であり；および
各Ｒ’は独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

Has the structure of
Where E is -O-, -S-, -NR'-or -C (R') _2- ;
D is = N-, = C (F)-, = C (Cl)-, = C (Br)-, = C (I)-, = C (CN)-, = C (NO ₂ )-, = C (CO _2- (C ₁ to C ₆ aliphatic))-or = C (CF ₃ )-; and each R'is independently defined as described above and as described herein. be.

の構造を有し、
ここで、
Ｇは、－Ｏ－、－Ｓ－、または－ＮＲ’であり；
Ｄは、＝Ｎ－、＝Ｃ（Ｆ）－、＝Ｃ（Ｃｌ）－、＝Ｃ（Ｂｒ）－、＝Ｃ（Ｉ）－、＝Ｃ（ＣＮ）－、＝Ｃ（ＮＯ_２）－、＝Ｃ（ＣＯ_２－（Ｃ_１～Ｃ_６脂肪族））－、または＝Ｃ（ＣＦ_３）－である。 In some embodiments, L is

Has the structure of
here,
G is -O-, -S-, or -NR';
D is = N-, = C (F)-, = C (Cl)-, = C (Br)-, = C (I)-, = C (CN)-, = C (NO ₂ )-, = C (CO _2- (C ₁ to C ₆ aliphatic))-or = C (CF ₃ )-.

の構造を有し、
ここで、
Ｅは、－Ｏ－、－Ｓ－、－ＮＲ’－又は－Ｃ（Ｒ’）_２－であり；
Ｄは、＝Ｎ－、＝Ｃ（Ｆ）－、＝Ｃ（Ｃｌ）－、＝Ｃ（Ｂｒ）－、＝Ｃ（Ｉ）－、＝Ｃ（ＣＮ）－、＝Ｃ（ＮＯ_２）－、＝Ｃ（ＣＯ_２－（Ｃ_１～Ｃ_６脂肪族））－、または＝Ｃ（ＣＦ_３）－であり；および
各Ｒ’は独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

Has the structure of
here,
E is -O-, -S-, -NR'-or -C (R') _2- ;
D is = N-, = C (F)-, = C (Cl)-, = C (Br)-, = C (I)-, = C (CN)-, = C (NO ₂ )-, = C (CO _2- (C ₁ to C ₆ aliphatic))-or = C (CF ₃ )-; and each R'is independently defined as described above and as described herein. be.

の構造を有し、
ここで、
Ｇは、－Ｏ－、－Ｓ－、または－ＮＲ’であり；
Ｄは、＝Ｎ－、＝Ｃ（Ｆ）－、＝Ｃ（Ｃｌ）－、＝Ｃ（Ｂｒ）－、＝Ｃ（Ｉ）－、＝Ｃ（ＣＮ）－、＝Ｃ（ＮＯ_２）－、＝Ｃ（ＣＯ_２－（Ｃ_１～Ｃ_６脂肪族））－、または＝Ｃ（ＣＦ_３）－である。 In some embodiments, L is

Has the structure of
here,
G is -O-, -S-, or -NR';
D is = N-, = C (F)-, = C (Cl)-, = C (Br)-, = C (I)-, = C (CN)-, = C (NO ₂ )-, = C (CO _2- (C ₁ to C ₆ aliphatic))-or = C (CF ₃ )-.

の構造を有し、
ここでＥは、－Ｏ－、－Ｓ－、－ＮＲ’－又は－Ｃ（Ｒ’）_２－であり；
- - -は単結合又は二重結合であり；
２つのＲ^Ｌ１は、これらが結合している２個の炭素原子と一緒になって、置換されていてもよいアリール、Ｃ_３～Ｃ_１０炭素環式、ヘテロアリール又はヘテロ環式環を形成し；
各Ｒ’は独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

Has the structure of
Where E is -O-, -S-, -NR'-or -C (R') _2- ;
---- is a single bond or a double bond;
The two ^RL1s together with the two carbon atoms to which they are attached form an optionally substituted aryl, C ₃ to C ₁₀ carbocyclic, heteroaryl or heterocyclic ring. ;
Each R'is independently defined as described above and is as described herein.

の構造を有し、
ここでＧは、－Ｏ－、－Ｓ－、または－ＮＲ’であり；
- - -は単結合又は二重結合であり；
２つのＲ^Ｌ１は、これらが結合している２個の炭素原子と一緒になって、置換されても良いアリール、Ｃ_３～Ｃ_１０炭素環式、ヘテロアリールまたはヘテロ環式環を形成し；
各Ｒ’は独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

Has the structure of
Where G is -O-, -S-, or -NR';
---- is a single bond or a double bond;
The two ^RL1s together with the two carbon atoms to which they are attached form an aryl, C ₃ to _C10 carbocyclic, heteroaryl or heterocyclic ring which may be substituted;
Each R'is independently defined as described above and is as described herein.

の構造を有し、
ここで、
Ｅは、－Ｏ－、－Ｓ－、－ＮＲ’－又は－Ｃ（Ｒ’）_２－であり；
Ｄは、＝Ｎ－、＝Ｃ（Ｆ）－、＝Ｃ（Ｃｌ）－、＝Ｃ（Ｂｒ）－、＝Ｃ（Ｉ）－、＝Ｃ（ＣＮ）－、＝Ｃ（ＮＯ_２）－、＝Ｃ（ＣＯ_２－（Ｃ_１～Ｃ_６脂肪族））－、または＝Ｃ（ＣＦ_３）－であり；および
各Ｒ’は独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

Has the structure of
here,
E is -O-, -S-, -NR'-or -C (R') _2- ;
D is = N-, = C (F)-, = C (Cl)-, = C (Br)-, = C (I)-, = C (CN)-, = C (NO ₂ )-, = C (CO _2- (C ₁ to C ₆ aliphatic))-or = C (CF ₃ )-; and each R'is independently defined as described above and as described herein. be.

の構造を有し、
ここでＧは、－Ｏ－、－Ｓ－、または－ＮＲ’であり；
Ｄは、＝Ｎ－、＝Ｃ（Ｆ）－、＝Ｃ（Ｃｌ）－、＝Ｃ（Ｂｒ）－、＝Ｃ（Ｉ）－、＝Ｃ（ＣＮ）－、＝Ｃ（ＮＯ_２）－、＝Ｃ（ＣＯ_２－（Ｃ_１～Ｃ_６脂肪族））－、または＝Ｃ（ＣＦ_３）－であり；および
各Ｒ’は独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

Has the structure of
Where G is -O-, -S-, or -NR';
D is = N-, = C (F)-, = C (Cl)-, = C (Br)-, = C (I)-, = C (CN)-, = C (NO ₂ )-, = C (CO _2- (C ₁ to C ₆ aliphatic))-or = C (CF ₃ )-; and each R'is independently defined as described above and as described herein. be.

の構造を有し、
ここでＥは、－Ｏ－、－Ｓ－、－ＮＲ’－又は－Ｃ（Ｒ’）_２－であり；
Ｄは、＝Ｎ－、＝Ｃ（Ｆ）－、＝Ｃ（Ｃｌ）－、＝Ｃ（Ｂｒ）－、＝Ｃ（Ｉ）－、＝Ｃ（ＣＮ）－、＝Ｃ（ＮＯ_２）－、＝Ｃ（ＣＯ_２－（Ｃ_１～Ｃ_６脂肪族））－、または＝Ｃ（ＣＦ_３）－であり；および
各Ｒ’は独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

Has the structure of
Where E is -O-, -S-, -NR'-or -C (R') _2- ;
D is = N-, = C (F)-, = C (Cl)-, = C (Br)-, = C (I)-, = C (CN)-, = C (NO ₂ )-, = C (CO _2- (C ₁ to C ₆ aliphatic))-or = C (CF ₃ )-; and each R'is independently defined as described above and as described herein. be.

の構造を有し、
ここでＧは、－Ｏ－、－Ｓ－、または－ＮＲ’であり；
Ｄは、＝Ｎ－、＝Ｃ（Ｆ）－、＝Ｃ（Ｃｌ）－、＝Ｃ（Ｂｒ）－、＝Ｃ（Ｉ）－、＝Ｃ（ＣＮ）－、＝Ｃ（ＮＯ_２）－、＝Ｃ（ＣＯ_２－（Ｃ_１～Ｃ_６脂肪族））－、または＝Ｃ（ＣＦ_３）－であり；および
各Ｒ’は独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

Has the structure of
Where G is -O-, -S-, or -NR';
D is = N-, = C (F)-, = C (Cl)-, = C (Br)-, = C (I)-, = C (CN)-, = C (NO ₂ )-, = C (CO _2- (C ₁ to C ₆ aliphatic))-or = C (CF ₃ )-; and each R'is independently defined as described above and as described herein. be.

の構造を有し、
ここでＥは、－Ｏ－、－Ｓ－、－ＮＲ’－又は－Ｃ（Ｒ’）_２－であり；
- - -は単結合又は二重結合であり；
２つのＲ^Ｌ１は、これらが結合している２個の炭素原子と一緒になって、置換されても良いアリール、Ｃ_３～Ｃ_１０炭素環式、ヘテロアリールまたはヘテロ環式環を形成し；
各Ｒ’は独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

Has the structure of
Where E is -O-, -S-, -NR'-or -C (R') _2- ;
---- is a single bond or a double bond;
The two ^RL1s together with the two carbon atoms to which they are attached form an aryl, C ₃ to _C10 carbocyclic, heteroaryl or heterocyclic ring which may be substituted;
Each R'is independently defined as described above and is as described herein.

の構造を有し、
ここでＧは、－Ｏ－、－Ｓ－、または－ＮＲ’であり；
- - -は単結合又は二重結合であり；
２つのＲ^Ｌ１は、これらが結合している２個の炭素原子と一緒になって、置換されても良いアリール、Ｃ_３～Ｃ_１０炭素環式、ヘテロアリールまたはヘテロ環式環を形成し；
各Ｒ’は独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

Has the structure of
Where G is -O-, -S-, or -NR';
---- is a single bond or a double bond;
The two ^RL1s together with the two carbon atoms to which they are attached form an aryl, C ₃ to _C10 carbocyclic, heteroaryl or heterocyclic ring which may be substituted;
Each R'is independently defined as described above and is as described herein.

の構造を有し、
ここでＥは、－Ｏ－、－Ｓ－、－ＮＲ’－又は－Ｃ（Ｒ’）_２－であり；
Ｄは、＝Ｎ－、＝Ｃ（Ｆ）－、＝Ｃ（Ｃｌ）－、＝Ｃ（Ｂｒ）－、＝Ｃ（Ｉ）－、＝Ｃ（ＣＮ）－、＝Ｃ（ＮＯ_２）－、＝Ｃ（ＣＯ_２－（Ｃ_１～Ｃ_６脂肪族））－、または＝Ｃ（ＣＦ_３）－であり；および
各Ｒ’は独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

Has the structure of
Where E is -O-, -S-, -NR'-or -C (R') _2- ;
D is = N-, = C (F)-, = C (Cl)-, = C (Br)-, = C (I)-, = C (CN)-, = C (NO ₂ )-, = C (CO _2- (C ₁ to C ₆ aliphatic))-or = C (CF ₃ )-; and each R'is independently defined as described above and as described herein. be.

の構造を有し、
ここで、
Ｇは、－Ｏ－、－Ｓ－、または－ＮＲ’であり；
Ｄは、＝Ｎ－、＝Ｃ（Ｆ）－、＝Ｃ（Ｃｌ）－、＝Ｃ（Ｂｒ）－、＝Ｃ（Ｉ）－、＝Ｃ（ＣＮ）－、＝Ｃ（ＮＯ_２）－、＝Ｃ（ＣＯ_２－（Ｃ_１～Ｃ_６脂肪族））－、または＝Ｃ（ＣＦ_３）－であり；および
Ｒ’は、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

Has the structure of
here,
G is -O-, -S-, or -NR';
D is = N-, = C (F)-, = C (Cl)-, = C (Br)-, = C (I)-, = C (CN)-, = C (NO ₂ )-, = C (CO _2- (C ₁ to C ₆ aliphatic))-or = C (CF ₃ )-; and R'as defined above and as described herein.

の構造を有し、
ここで、
Ｅは、－Ｏ－、－Ｓ－、－ＮＲ’－又は－Ｃ（Ｒ’）_２－であり；
Ｄは、＝Ｎ－、＝Ｃ（Ｆ）－、＝Ｃ（Ｃｌ）－、＝Ｃ（Ｂｒ）－、＝Ｃ（Ｉ）－、＝Ｃ（ＣＮ）－、＝Ｃ（ＮＯ_２）－、＝Ｃ（ＣＯ_２－（Ｃ_１～Ｃ_６脂肪族））－、または＝Ｃ（ＣＦ_３）－であり；および
各Ｒ’は独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

Has the structure of
here,
E is -O-, -S-, -NR'-or -C (R') _2- ;
D is = N-, = C (F)-, = C (Cl)-, = C (Br)-, = C (I)-, = C (CN)-, = C (NO ₂ )-, = C (CO _2- (C ₁ to C ₆ aliphatic))-or = C (CF ₃ )-; and each R'is independently defined as described above and as described herein. be.

の構造を有し、
ここで、
Ｇは、－Ｏ－、－Ｓ－、または－ＮＲ’であり；
Ｄは、＝Ｎ－、＝Ｃ（Ｆ）－、＝Ｃ（Ｃｌ）－、＝Ｃ（Ｂｒ）－、＝Ｃ（Ｉ）－、＝Ｃ（ＣＮ）－、＝Ｃ（ＮＯ_２）－、＝Ｃ（ＣＯ_２－（Ｃ_１～Ｃ_６脂肪族））－、または＝Ｃ（ＣＦ_３）－であり；および
Ｒ’は、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

Has the structure of
here,
G is -O-, -S-, or -NR';
D is = N-, = C (F)-, = C (Cl)-, = C (Br)-, = C (I)-, = C (CN)-, = C (NO ₂ )-, = C (CO _2- (C ₁ to C ₆ aliphatic))-or = C (CF ₃ )-; and R'as defined above and as described herein.

の構造を有し、
ここで、フェニル環は、置換されていてもよい。いくつかの実施形態において、フェニル環は、置換されない。いくつかの実施形態において、フェニル環は、置換される。 In some embodiments, L is

Has the structure of
Here, the phenyl ring may be substituted. In some embodiments, the phenyl ring is not substituted. In some embodiments, the phenyl ring is substituted.

の構造を有し、
ここで、フェニル環は、置換されていてもよい。いくつかの実施形態において、フェニル環は、置換されない。いくつかの実施形態において、フェニル環は、置換される。 In some embodiments, L is

Has the structure of
Here, the phenyl ring may be substituted. In some embodiments, the phenyl ring is not substituted. In some embodiments, the phenyl ring is substituted.

の構造を有し、
ここで- - -は単結合又は二重結合であり；および
２つのＲ^Ｌ１は、これらが結合している２個の炭素原子と一緒になって、置換されていてもよいアリール、Ｃ_３～Ｃ_１０炭素環式、ヘテロアリール又はヘテロ環式環を形成する。 In some embodiments, L is

Has the structure of
Where --- is a single bond or a double bond; and the two ^RL1s may be substituted together with the two carbon atoms to which they are attached aryl _, C3 ~. Form a _C10 carbocyclic, heteroaryl or heterocyclic ring.

の構造を有し、
ここで、
Ｇは、－Ｏ－、－Ｓ－、または－ＮＲ’であり；
- - -は単結合又は二重結合であり；および
２つのＲ^Ｌ１は、これらが結合している２個の炭素原子と一緒になって、置換されても良いアリール、Ｃ_３～Ｃ_１０炭素環式、ヘテロアリールまたはヘテロ環式環を形成する。 In some embodiments, L is

Has the structure of
here,
G is -O-, -S-, or -NR';
---- are single or double bonds; and the two ^RL1s may be substituted with the two carbon atoms to which they are attached aryl, C ₃ to C ₁₀ carbons. Form a cyclic, heteroaryl or heterocyclic ring.

Generally, as described above and herein, E is -O-, -S-, -NR'-or -C (R') _2- , and each R'is independently described above. As defined and as described here. In some embodiments, E is -O-, -S-, or -NR'-. In some embodiments, E is —O—, —S—, or —NH—. In some embodiments, E is —O—. In some embodiments, E is —S—. In some embodiments, E is -NH-.

Generally, as described above and herein, G is -O-, -S-, or -NR', and each R'is independently defined as above and as described herein. Is. In some embodiments, G is —O—, —S—, or —NH—. In some embodiments, G is —O—. In some embodiments, G is —S—. In some embodiments, G is -NH-.

により置換され、
ＧおよびＲ’のそれぞれおよび環Ｃｙ’は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is -L ³ _- G-, where L ³ is optionally substituted C _1-5 alkylene or alkenylene, with one or more methylene units being optional. -O-, -S-, -N (R')-, -C (O)-, -C (S)-, -C (NR')-, -S (O)-, independently -S (O) ₂ -or

Replaced by
Each of G and R'and the ring Cy'are independently defined as described above and are as described herein.

In some embodiments, L is —L ³ -S— and L ³ is as defined above and as described herein. In some embodiments, L is —L ³ -O— and L ³ is as defined above and as described herein. In some embodiments, L is -L ³ -N (R')-where each of L ³ and R'is independently defined as described above and as described herein. be. In some embodiments, L is -L ³ -NH-, and each of L ³ and R'is independently defined as described above and is as described herein.

により置換され、Ｒ’および環Ｃｙ’のそれぞれは、独立して、上記のとおり定義し、ここに記載したとおりである。いくつかの実施形態において、Ｌ^３は、置換されていてもよいＣ_５アルキレンである。いくつかの実施形態において、－Ｌ^３－Ｇ－は、

である。 _In some embodiments, L ³ is a C5 alkylene or alkenylene which may be substituted, with one or more methylene units being arbitrarily and independently -O-, -S-, -N (R). ´)-, -C (O)-, -C (S)-, -C (NR')-, -S (O)-, -S (O) _2- , or

Substituted by, each of R'and Ring Cy'is independently defined as described above and is as described herein. ^In some embodiments, L3 is a _C5 alkylene that may be substituted. In some embodiments, -L ³ -G-

Is.

により置換され、Ｒ’およびＣｙ’のそれぞれは、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L ³ is _C4 alkylene or alkenylene which may be substituted, and one or more methylene units are optionally and independently -O-, -S-, -N (R). ´)-, -C (O)-, -C (S)-, -C (NR')-, -S (O)-, -S (O) _2- , or

Substituted by, each of R'and Cy'is independently defined as described above and is as described herein.

である。 In some embodiments, -L ³ -G-

Is.

により置換され、Ｒ’およびＣｙ’のそれぞれは、独立して、上記のとおり定義し、ここに記載したとおりである。 ^In some embodiments _, L3 is a C3 alkylene or alkenylene which may be substituted, with one or more methylene units being optionally and independently -O-, -S-, -N (R). ´)-, -C (O)-, -C (S)-, -C (NR')-, -S (O)-, -S (O) _2- , or

Substituted by, each of R'and Cy'is independently defined as described above and is as described herein.

である。 In some embodiments, -L ³ -G-

Is.

である。いくつかの実施形態において、Ｌは、

である。いくつかの実施形態において、Ｌは、

である。 In some embodiments, L is

Is. In some embodiments, L is

Is. In some embodiments, L is

Is.

により任意におよび独立して置換され、Ｒ’およびＣｙ’のそれぞれは、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L ³ may be substituted C ₂ is alkylene or alkenylene and one or more methylene units are -O-, -S-, -N (R')-, -C (O)-, -C (S)-, -C (NR')-, -S (O)-, -S (O) _2- , or

Arbitrarily and independently substituted by, each of R'and Cy'is independently defined as described above and is as described herein.

であり、ＧおよびＣｙ’のそれぞれは、独立して、上記のとおり定義し、ここに記載したとおりである。いくつかの実施形態において、Ｌは、

である。 In some embodiments, -L ³ -G-

And each of G and Cy'is independently defined as described above and is as described herein. In some embodiments, L is

Is.

In some embodiments, L is -L ⁴ -G-, L ⁴ is a optionally substituted C ₁ to C ₂ alkylene; and G is defined and herein as described above. As described. In some embodiments, L is -L ⁴ -G- and L ⁴ is a optionally substituted C ₁ to C ₂ alkylene; G is defined and described herein as described above. As you did; G is bound to ^R1 . In some embodiments, L is -L ⁴ -G- and L ⁴ is methylene that may be substituted; G is as defined above and as described herein; And G are bound to ^R1 . In some embodiments, L is -L ⁴ -G- and L ⁴ is methylene; G is defined above and is as described herein; and G is R ¹ Is combined with. In some embodiments, L is -L ⁴ -G- and L ⁴ may be substituted- (CH ₂ ) _2- ; G is defined and herein as described above. As described; and G is associated with ^R1 . In some embodiments, L is -L ⁴ -G- and L ⁴ is-(CH ₂ ) _2- ; G is defined above and is as described herein; And G are bound to ^R1 .

または

であり、ここで、Ｇは、上記のとおり定義し、ここに記載したとおりであり、Ｇは、Ｒ^１に結合されている。いくつかの実施形態において、Ｌは、

であり、Ｇは、上記のとおり定義し、ここに記載したとおりであり、Ｇは、Ｒ^１に結合されている。いくつかの実施形態において、Ｌは、

であり、Ｇは、上記のとおり定義し、ここに記載したとおりであり、Ｇは、Ｒ^１に結合されている。いくつかの実施形態において、Ｌは、

または

であり、ここで硫黄原子は、Ｒ^１に結合されている。いくつかの実施形態において、Ｌは、

または

であり、ここで酸素原子は、Ｒ^１に結合されている。 In some embodiments, L is

or

Here, G is defined as described above and is as described herein, and G is bound to ^R1 . In some embodiments, L is

G is defined above and is as described herein, where G is bound to ^R1 . In some embodiments, L is

G is defined above and is as described herein, where G is bound to ^R1 . In some embodiments, L is

or

Here, the sulfur atom is bonded to ^R1 . In some embodiments, L is

or

Here, the oxygen atom is bonded to ^R1 .

であり、Ｇは、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

And G is defined as described above and is as described herein.

In some embodiments, L is -S-R ^L3- or -SC (O) -R ^L3- , where ^RL3 is optionally substituted and linear or branched C ₁ -C ₉ It is alkylene, and one or more methylene units may be substituted C ₁ to C ₆ alkylene, C ₁ to C ₆ alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R')-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-,-S (O)-,-S (O) _2- , -S (O) ₂ N (R')-,-N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O)-, or -C (O) O-, optionally and independently, where R'and -Cy- Each of these is independently defined as described above and is as described herein. In some embodiments, L is -S-R ^L3- or -SC (O) -R ^L3- and ^RL3 is a C ₁ to C ₆ alkylene that may be substituted. In some embodiments, L is -S-R ^L3- or -SC (O) -R ^L3- and ^RL3 is C ₁ to C ₆ alkenylene which may be substituted. In some embodiments, L is -S-R ^L3- or -SC (O) -R ^L3- and ^RL3 is a optionally substituted C ₁ to C ₆ alkylene. _One or more methylene units are optionally and independently substituted with _C1-6 alkenylene, arylene, or heteroarylene which may be substituted. In some embodiments, in some embodiments, ^RL3 may be substituted-S- (C ₁ to C ₆ alkenylene)-, -S- (C ₁ to C ₆ alkylene)-, -S- (C ₁ to C ₆ alkylene) -allylen- (C _{1 to C 6 alkylene)-, -S-CO-allylene- (C 1 to C 6 alkylene )} -, or -S-CO- (C ₁ ) -C ₆ alkylene) -allylene- (C ₁ to C ₆ alkylene)-.

である。 In some embodiments, L is

Is.

である。
いくつかの実施形態において、Ｌは、

である。
いくつかの実施形態において、

である。 In some embodiments, L is

Is.
In some embodiments, L is

Is.
In some embodiments

Is.

In some embodiments, the sulfur atom of L in the above and in the embodiments described herein is attached to X. In some embodiments, the sulfur atom of L in the above and in the embodiments described herein is attached to ^R1 .

Generally, as described above and herein, R ¹ is a halogen, R, or optionally substituted C ₁ to C ₅₀ aliphatic, and one or more methylene units may be substituted. C ₁ to C ₆ alkylene, C ₁ to C ₆ alkaneylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N ( R')-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')-,-N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-,-S (O)-, -S (O) _2- , -S (O) ₂ N (R')-, -N (R') S (O) _2- , -SC (O)-, -C (O) S-,- Replaced arbitrarily and independently by OC (O)-or-C (O) O-where each variable is independently defined as described above and as described herein. In some embodiments, R ¹ is a halogen, R, or optionally substituted C ₁ to C ₁₀ aliphatic, and one or more methylene units may be substituted C ₁ to C ₆ Alkylene, C ₁ to C ₆ alkaneylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -SS-, -N (R')-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')- , -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-, -S (O) _2- , -S (O) ₂ N (R')-,-N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O)- , Or -C (O) O-, optionally and independently, where each variable is independently defined as described above and as described herein.

In some embodiments, R ¹ is hydrogen. In some embodiments, R ¹ is a halogen. In some embodiments, R ¹ is −F. In some embodiments, R ¹ is -Cl. In some embodiments, R ¹ is -Br. In some embodiments, R ¹ is -I.

In some embodiments, R ¹ is R, where R is defined above and is as described herein.

In some embodiments, R ¹ is hydrogen. In some embodiments, R ¹ is a optionally substituted group selected from C ₁ to C ₅₀ aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl.

In some embodiments, R ¹ is an optionally substituted C ₁ to C ₅₀ aliphatic. In some embodiments, R ¹ is an optionally substituted C ₁ to C ₁₀ aliphatic. In some embodiments, R ¹ is an optionally substituted C ₁ to C ₆ aliphatic. In some embodiments, R ¹ is a C ₁ to C ₆ alkyl which may be substituted. In some embodiments, R ¹ is an optionally substituted, linear or branched hexyl. In some embodiments, R ¹ is optionally substituted, linear or branched pentyl. In some embodiments, R ¹ is optionally substituted, linear or branched butyl. In some embodiments, R ¹ is optionally substituted, linear or branched propyl. In some embodiments, R ¹ is an ethyl that may be substituted. In some embodiments, R ¹ is a methyl that may be substituted.

In some embodiments, R ¹ is a optionally substituted phenyl. In some embodiments, R ¹ is a substituted phenyl. In some embodiments, R ¹ is phenyl.

In some embodiments, R ¹ is a carbocyclyl that may be substituted. In some embodiments, R ¹ is a C ₃ to C ₁₀ carbocyclyl that may be substituted. In some embodiments, R ¹ is a monocyclic carbocyclyl that may be substituted. In some embodiments, R ¹ is a optionally substituted cycloheptyl. In some embodiments, R ¹ is a cyclohexyl that may be substituted. In some embodiments, R ¹ is cyclopentyl which may be substituted. In some embodiments, R ¹ is a optionally substituted cyclobutyl. In some embodiments, R ¹ is cyclopropyl which may be substituted. In some embodiments, R ¹ is a bicyclic carbocyclyl that may be substituted.

である。いくつかの実施形態において、Ｒ^１は、

である。いくつかの実施形態において、Ｒ^１は、置換されていてもよい

である。 In some embodiments, R ¹ is a C ₁ to C ₅₀ polycyclic hydrocarbon that may be substituted. In some embodiments, R ¹ is a optionally substituted C ₁ to C ₅₀ polycyclic hydrocarbon and one or more methylene units are optionally substituted C ₁ to C ₆ alkylene, C ₁ to C ₆ Alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R')-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')-,- N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-, -S (O) _2- , -S (O) ₂ N (R')-, -N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O)-, or Arbitrarily and independently substituted by —C (O) O—, where each variable is independently defined as described above and as described herein. In some embodiments, R ¹ may be substituted.

Is. In some embodiments, R ¹ is

Is. In some embodiments, R ¹ may be substituted.

Is.

を含む。いくつかの実施形態において、Ｒ^１は、

である。いくつかの実施形態において、Ｒ^１は、

である。いくつかの実施形態において、Ｒ^１は、

である。いくつかの実施形態において、Ｒ^１は、

である。いくつかの実施形態において、Ｒ^１は、

である。 In some embodiments, R ¹ is an optionally substituted C ₁ to C ₅₀ aliphatic comprising one or more optionally substituted polycyclic hydrocarbon moieties. In some embodiments, R ¹ is an optionally substituted C ₁ to C ₅₀ aliphatic comprising one or more optionally substituted polycyclic hydrocarbon moieties and one or more methylene units. May be substituted C ₁ to C ₆ alkylene, C ₁ to C ₆ alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-,- SS-, -N (R')-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N ( R') C (O) N (R')-,-N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')- , -S (O)-,-S (O) _2- , -S (O) ₂ N (R')-,-N (R') S (O) _2- , -SC (O)-,- Arbitrarily and independently substituted by C (O) S-, -OC (O)-, or -C (O) O-, where each variable is independently defined as described above and here. As described in. In some embodiments, R ¹ is one or more, optionally substituted C ₁ to C ₅₀ aliphatics.

including. In some embodiments, R ¹ is

Is. In some embodiments, R ¹ is

Is. In some embodiments, R ¹ is

Is. In some embodiments, R ¹ is

Is. In some embodiments, R ¹ is

Is.

In some embodiments, R ¹ is an optionally substituted aryl. In some embodiments, R ¹ is a optionally substituted bicyclic aryl ring.

In some embodiments, R ¹ is a heteroaryl which may be substituted. In some embodiments, R ¹ is a 5- to 6-membered monocyclic heteroaryl ring that may be substituted with 1 to 3 heteroatoms independently selected from nitrogen, sulfur, or oxygen. be. In some embodiments, R ¹ is a substituted 5- to 6-membered monocyclic heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R ¹ is an unsubstituted 5- to 6-membered monocyclic heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, sulfur, or oxygen.

In some embodiments, R ¹ is a optionally substituted 5-membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen or sulfur. In some embodiments, R ¹ is a optionally substituted 6-membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R ¹ is a optionally substituted 5-membered monocyclic heteroaryl ring having one heteroatom selected from nitrogen, oxygen or sulfur. In some embodiments, R ¹ is selected from pyrrolyl, furanyl, or thienyl.

In some embodiments, R ¹ is a optionally substituted 5-membered heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R ¹ is a optionally substituted 5-membered heteroaryl ring having one nitrogen atom, the additional heteroatom being selected from sulfur or oxygen. ^An exemplary R group comprises substituted pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl or isooxazolyl.

In some embodiments, R ¹ is a 6-membered heteroaryl ring with 1-3 nitrogen atoms. In another embodiment, R ¹ is a optionally substituted 6-membered heteroaryl ring having 1-2 nitrogen atoms. In some embodiments, R ¹ is a optionally substituted 6-membered heteroaryl ring having two nitrogen atoms. In certain embodiments, R ¹ is a optionally substituted 6-membered heteroaryl ring having one nitrogen. Exemplary R groups are pyridinyl, pyrimidinyl, pyrazinyl, pyridadinyl, triazinyl, or tetrazinyl which may be substituted.

In certain embodiments, R ¹ is an 8- to 10-membered bicyclic heteroaryl ring that may be substituted with 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. be. In some embodiments, R ¹ is a optionally substituted 5,6-condensed heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In another embodiment, R ¹ is an optionally substituted 5,6-condensed heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R ¹ is a optionally substituted 5,6-condensed heteroaryl ring having one heteroatom independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R ¹ is an indrill that may be substituted. In some embodiments, R ¹ is an optionally substituted azabicyclo [3.2.1] octanyl. In certain embodiments, R ¹ is an optionally substituted 5,6-condensed heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R ¹ is an azaindolyl that may be substituted. In some embodiments, R ¹ is a benzimidazolyl that may be substituted. In some embodiments, R ¹ is a optionally substituted benzothiazolyl. In some embodiments, R ¹ is a optionally substituted benzoxazolyl. In some embodiments, R ¹ is an indazolyl that may be substituted. In certain embodiments, R ¹ is an optionally substituted 5,6-condensed heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, R ¹ is a optionally substituted 6,6-condensed heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R ¹ is a optionally substituted 6,6-condensed heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In another embodiment, R ¹ is a optionally substituted 6,6-condensed heteroaryl ring having one heteroatom independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R ¹ is a optionally substituted quinolinyl. In some embodiments, R ¹ is an isoquinolinyl that may be substituted. According to some embodiments, R ¹ is an optionally substituted 6,6-condensed heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R ¹ is quinazoline or quinoxaline.

In some embodiments, R ¹ is a heterocyclyl that may be substituted. In some embodiments, R ¹ is 3- to 7-membered saturated or partially unsaturated, which may be substituted with 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. It is a heterocyclic ring of. In some embodiments, R ¹ is a substituted 3- to 7-membered saturated or partially unsaturated heterocycle with 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. It is a formula ring. In some embodiments, R ¹ is an unsaturated 3- to 7-membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. It is a formula ring.

In some embodiments, R ¹ is a heterocyclyl that may be substituted. In some embodiments, R ¹ is a 6-membered saturated or partially unsaturated hetero that may be substituted with 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. It is a cyclic ring. In some embodiments, R ¹ is a partially unsaturated 6-membered heterocyclic ring that may be substituted with two heteroatoms independently selected from nitrogen, oxygen, or sulfur. be. In some embodiments, R ¹ is a optionally substituted, partially unsaturated 6-membered heterocyclic ring having two oxygen atoms.

In certain embodiments, R ¹ is a 3- to 7-membered saturated or partially unsaturated heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. be. In certain embodiments, R ¹ is oxylanyl, oxetanyl, tetrahydropyranyl, tetrahydropyranyl, oxepanyl, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepanyl, tiylanyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, thiepanyl, dioxolanyl. , Oxazolidine, oxazolidinyl, imidazolidinyl, thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl, piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl, oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl Thiophenonyl, tetrahydrothiopyranonyl, thiepanonyl, oxazolidinonyl, oxadinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl, dioxepanonyl, oxathiolinonyl, oxathianonyl, oxathiepannyl, thiazolidinonyl, thiazinanoyl, thiazepanonyl, imidazoline. Nonyl, diazepanonyl, imidazolidinedionyl, oxazolidinezionyl, thiazolidinezonyl, dioxolandionyl, oxathiolandionyl, piperazinezionyl, morpholindionyl, thiomorpholinzionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl , Piperidinyl, piperazinyl, pyrrolidinyl, tetrahydrothiophenyl, or tetrahydrothiopyranyl. In some embodiments, R is a 5-membered saturated or partially unsaturated heterocycle that may be substituted with 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. It is a formula ring. In some embodiments, R ¹ is a 5-membered saturated or partially unsaturated hetero that may be substituted with 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. It is a cyclic ring.

In certain embodiments, R ¹ may be substituted with 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. 5-6 members Partially unsaturated single It is a cyclic ring. In certain embodiments, R ¹ is a optionally substituted tetrahydropyridinyl, dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

In some embodiments, R ¹ is optionally substituted with 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 8- to 10-membered bicyclic saturated or partially saturated. It is an unsaturated heterocyclic ring. In some embodiments, R ¹ is an indolinyl that may be substituted. In some embodiments, R ¹ is an isoindrinyl that may be substituted. In some embodiments, R ¹ is 1,2,3,4-tetrahydroquinoline which may be substituted. In some embodiments, R is 1,2,3,4-tetrahydroisoquinoline which may be substituted.

In some embodiments, R ¹ is an optionally substituted C ₁ to C ₁₀ aliphatic and one or more methylene units may be substituted C ₁ to C ₆ alkylene, C ₁ to. C ₆ Alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R')-, -C (O) -, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')-, -N (R) ´) C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-, -S (O) _2- , -S (O) ₂ N (R')-,-N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O)-, or -C ( O) Arbitrarily and independently substituted by O-, where each variable is independently defined as described above and as described herein. In some embodiments, R ¹ is an optionally substituted C ₁ to C ₁₀ aliphatic and one or more methylene units may be substituted —Cy—, —O—, —S. -, -S-S-, -N (R')-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R) ´)-, -S (O)-, -S (O) _2- , -S (O) ₂ N (R')-, -N (R') S (O) _2- , -OC (O) Arbitrarily and independently substituted by-or-C (O) O-, each R'is independently defined as described above and is as described herein. In some embodiments, R ¹ is an optionally substituted C ₁ to C ₁₀ aliphatic and one or more methylene units are any -Cy-, -O-, -S-, -S. Arbitrarily and independently replaced by -S-, -N (R')-, -C (O)-, -OC (O)-, or -C (O) O-, each R'is independent. Then, as defined above, it is as described here.

である。 In some embodiments, R ¹ is

Is.

である。 In some embodiments, R ¹ is

Is.

。 In some embodiments, R ¹ comprises a terminal having a optionally substituted- (CH ₂ ) ₂ -part bound to L. ^One such exemplary R is:

..

。 In some embodiments, R ¹ is bound to L and comprises a optionally substituted-(CH ₂ ) -part. ^One such exemplary R is:

..

In some embodiments, R ¹ is —S—R ^L2 , ^RL2 is C ₁ to C ₉ aliphatic, which may be substituted, and one or more methylene units are substituted. C ₁ to C ₆ alkylene, C ₁ to C ₆ alkaneylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S. -, -N (R')-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-,-S (O)-,-S (O) _2- , -S (O) ₂ N (R')-, -N (R') S (O) _2- , -SC (O)-, -C (O) ) S-, -OC (O)-, or -C (O) O-, optionally and independently, each of R'and -Cy- are independently defined as described above and herein. As described in. In some embodiments, R ¹ is —S—R ^L2 and the sulfur atom is bonded to the sulfur atom of the L group.

In some embodiments, R ¹ is -C (O) ^-RL 2 and ^RL 2 is an optionally substituted C ₁ to C ₉ aliphatic, with one or more methylene units being substituted. C ₁ to C ₆ alkylene, C ₁ to C ₆ alkaneylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S. -, -N (R')-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-,-S (O)-,-S (O) _2- , -S (O) ₂ N (R')-, -N (R') S (O) _2- , -SC (O)-, -C (O) ) S-, -OC (O)-, or -C (O) O-, optionally and independently, each of R'and -Cy- are independently defined as described above and herein. As described in. In some embodiments, R ¹ is -C (O) -RL 2 and the carbonyl group is attached to G of the ^L group. In some embodiments, R ¹ is -C (O) -RL 2 and the carbonyl group is attached to the sulfur atom of the ^L group.

In some embodiments, ^RL2 is an optionally substituted C ₁ to C ₉ aliphatic. In some embodiments, ^RL2 is a _{optionally substituted C 1-9 alkyl} . In some embodiments, ^RL2 is a _optionally substituted C _1-9 alkenyl. In some embodiments, ^RL2 is a _optionally substituted C _1-9 alkynyl. In some embodiments, ^RL2 is a C ₁ to C ₉ aliphatic that may be substituted, with one or more methylene units being optionally and independently -Cy- or -C (O)-. Is replaced by. In some embodiments, ^RL2 is a C ₁ to C ₉ aliphatic which may be substituted, and one or more methylene units are optionally and independently substituted by —Cy—. In some embodiments, ^RL2 is an optionally substituted C ₁ to C ₉ aliphatic, and one or more methylene units are optionally and independently substituted with optionally substituted heterosilene. To. In some embodiments, ^RL2 is an optionally substituted C ₁ to C ₉ aliphatic, and one or more methylene units are optionally and independently substituted with optionally substituted arylene. To. In some embodiments, ^RL2 is an optionally substituted C ₁ to C ₉ aliphatic, and the above methylene units are optionally and independently substituted with optionally substituted heteroarylene. To. In some embodiments, ^RL2 is an optionally substituted C ₁ to C ₉ aliphatic and one or more methylene units are optionally substituted with C ₃ to C ₁₀ carbocyclylene. And independently replaced. In some embodiments, ^RL2 is a C ₁ to C ₉ aliphatic which may be substituted, and the two methylene units are optionally and independently by -Cy- or -C (O)-. Will be replaced. In some embodiments, ^RL2 is a C ₁ to C ₉ aliphatic which may be substituted, and the two methylene units are optionally and independently by -Cy- or -C (O)-. Will be replaced. Exemplary ^RL2 groups are:

－Ｓ－（Ｃ_１～Ｃ_１０脂肪族）、Ｃ_１～Ｃ_１０脂肪族、アリール、Ｃ_１～Ｃ_６ヘテロアルキル、ヘテロアリールおよびヘテロシクリルから選択される。いくつかの実施形態において、Ｒ^１は、

または－Ｓ－（Ｃ_１～Ｃ_１０脂肪族）である。いくつかの実施形態において、Ｒ^１は、

である。 In some embodiments, R ¹ is a hydrogen or optionally substituted group.

-S- (C ₁ to C ₁₀ aliphatic), C ₁ to C ₁₀ aliphatic, aryl, C ₁ to C ₆ heteroalkyl, heteroaryl and heterocyclyl are selected. In some embodiments, R ¹ is

Or -S- (C ₁ to C ₁₀ aliphatic). In some embodiments, R ¹ is

Is.

In some embodiments, R ¹ is selected from —S— (C ₁ to C ₆ aliphatic), C ₁ to C ₁₀ aliphatic, C ₁ to C ₆ heteroaliphatic, aryl, heterocyclyl and heteroaryl. It is a group that may be substituted.

である。 In some embodiments, R ¹ is

Is.

In some embodiments, the sulfur atom of the embodiment of ^R1 described above and herein is in the sulfur atom, G, E, or —C (O) —part of the embodiment of L described above and herein. It is combined. In some embodiments, the -C (O) -part of the embodiment of R ¹ described above and herein is a sulfur atom, G, E, or -C (of the embodiments L described above and herein). O) -It is combined with the part.

In some embodiments, -L-R ¹ is one of the embodiments of L and the combination of embodiments of R ¹ described above and herein.

In some embodiments, -L-R ¹ is -L ³ -GR ¹ , where each variable is independently defined as described above and as described herein.

In some embodiments, -L-R ¹ is -L ⁴ -GR ¹ , where each variable is independently defined as described above and as described herein.

In some embodiments, -L-R ¹ is -L ³ -GS-R ^L2 , where each variable is independently defined as described above and as described herein. be.

In some embodiments, -L-R ¹ is -L ³ -GC (O) ^-RL 2, where each variable is independently defined and described above. As you did.

であり、ここでＲ^Ｌ２は、置換されていてもよいＣ_１～Ｃ_９脂肪族であり、１以上のメチレン単位は、置換されていてもよいＣ_１～Ｃ_６アルキレン、Ｃ_１～Ｃ_６アルケニレン、－Ｃ≡Ｃ－、－Ｃ（Ｒ´）_２－、－Ｃｙ－、－Ｏ－、－Ｓ－、－Ｓ－Ｓ－、－Ｎ（Ｒ´）－、－Ｃ（Ｏ）－、－Ｃ（Ｓ）－、－Ｃ（ＮＲ´）－、－Ｃ（Ｏ）Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｃ（Ｏ）Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｃ（Ｏ）－、－Ｎ（Ｒ´）Ｃ（Ｏ）Ｏ－、－ＯＣ（Ｏ）Ｎ（Ｒ´）－、－Ｓ（Ｏ）－、－Ｓ（Ｏ）_２－、－Ｓ（Ｏ）_２Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｓ（Ｏ）_２－、－ＳＣ（Ｏ）－、－Ｃ（Ｏ）Ｓ－、－ＯＣ（Ｏ）－、または－Ｃ（Ｏ）Ｏ－により任意におよび独立して置換され、各Ｇは、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Here, ^RL2 is an optionally substituted C ₁ to C ₉ aliphatic, and one or more methylene units may be substituted C ₁ to C ₆ alkylene, C ₁ to C ₆ Alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R')-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-, -S (O) _2- , -S (O) ) ₂ N (R')-,-N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O)-, or -C (O) Arbitrarily and independently substituted by O-, each G is independently defined as described above and as described herein.

In some embodiments, -L-R ¹ is -R ^L3 -S-S-R ^L2 , where each variable is independently defined as described above and as described herein. be. In some embodiments, -L-R ¹ is -R ^L3 -C (O) -S-S-R ^L2 , where each variable is independently defined as described above and here. As described in.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, -LR ¹ is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, L is

Has the structure of
Here, each variable is independently defined as described above and is as described herein.

の構造を有し、
ここでフェニル環は、置換されていてもよく、および
Ｒ^１およびＸのそれぞれは、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, the -X-L-R ¹ is

Has the structure of
Here the phenyl ring may be substituted, and each of ^R1 and X is independently defined as described above and as described herein.

である。 In some embodiments, -LR ¹ is

Is.

である。 In some embodiments, -LR ¹ is

Is.

である。いくつかの実施形態において、－Ｌ－Ｒ^１は、

である。 In some embodiments, -LR ¹ is

Is. In some embodiments, -LR ¹ is

Is.

。 In some embodiments, -L-R ¹ comprises the end of an X-bound, optionally substituted- (CH ₂ ) ₂ -part. In some embodiments, -L-R ¹ comprises the end of the-(CH ₂ ) ₂ -part bound to X. One such exemplary -LR ¹ part is as follows:

..

。 In some embodiments, -L-R ¹ comprises the end of an X-bound, optionally substituted- (CH ₂ ) -part. In some embodiments, -L-R ¹ comprises the end of the-(CH ₂ ) -part bound to X. One such exemplary -LR ¹ part is as follows:

..

である。 In some embodiments, -LR ¹ is

Is.

であり；およびＸは、－Ｓ－である。 In some embodiments, -LR ¹ is

And X is -S-.

であり、Ｘは、－Ｓ－、Ｗは、Ｏ、Ｙは、－Ｏ－、およびＺは、－Ｏ－である。 In some embodiments, -LR ¹ is

X is -S-, W is O, Y is -O-, and Z is -O-.

または－Ｓ－（Ｃ_１～Ｃ_１０脂肪族）である。 In some embodiments, R ¹ is

Or -S- (C ₁ to C ₁₀ aliphatic).

である。 In some embodiments, R ¹ is

Is.

または－Ｓ－（Ｃ_１～Ｃ_１０脂肪族）である。 In some embodiments, X is -O- or -S-, and R ¹ is.

Or -S- (C ₁ to C ₁₀ aliphatic).

－Ｓ－（Ｃ_１～Ｃ_１０脂肪族）または－Ｓ－（Ｃ_１～Ｃ_５０脂肪族）である。 In some embodiments, X is -O- or -S-, and R ¹ is.

-S- (C ₁ to C ₁₀ aliphatic) or -S- (C ₁ to C ₅₀ aliphatic).

In some embodiments, L is a covalent bond and —L—R ¹ is R ¹ .

In some embodiments, -LR ¹ is non-hydrogen.

、－Ｓ－（Ｃ_１～Ｃ_１０脂肪族）または－Ｓ－（Ｃ_１～Ｃ_５０脂肪族）である。 In some embodiments, -X-L-R ¹ is R ¹ is

, -S- (C ₁ to C ₁₀ aliphatic) or -S- (C ₁ to C ₅₀ aliphatic).

の構造を有し、部分

は、置換されていてもよい。いくつかの実施形態において、－Ｘ－Ｌ－Ｒ^１は、

である。いくつかの実施形態において、－Ｘ－Ｌ－Ｒ^１は、

である。いくつかの実施形態において、－Ｘ－Ｌ－Ｒ^１は、

である。いくつかの実施形態において、－Ｘ－Ｌ－Ｒ^１は、

の構造を有し、Ｘ’は、ＯまたはＳであり、Ｙ’は、－Ｏ－であり、－Ｓ－または－ＮＲ’－であり、部分

は、置換されていてもよい。いくつかの実施形態において、Ｙ’は、－Ｏ－であり、－Ｓ－または－ＮＨ－である。いくつかの実施形態において、

は、

である。いくつかの実施形態において、

は、

である。いくつかの実施形態において、

は、

である。いくつかの実施形態において、－Ｘ－Ｌ－Ｒ^１は、

の構造を有し、Ｘ’は、ＯまたはＳであり、部分

は、置換されていてもよい。いくつかの実施形態において、

は、

である。いくつかの実施形態において、－Ｘ－Ｌ－Ｒ^１は、

であり、

は、置換されていてもよい。いくつかの実施形態において、－Ｘ－Ｌ－Ｒ^１は、

であり、

は、置換される。いくつかの実施形態において、－Ｘ－Ｌ－Ｒ^１は、

であり、

は、非置換である。 In some embodiments, the -X-L-R ¹ is

Has the structure of, part

May be replaced. In some embodiments, the -X-L-R ¹ is

Is. In some embodiments, the -X-L-R ¹ is

Is. In some embodiments, the -X-L-R ¹ is

Is. In some embodiments, the -X-L-R ¹ is

X'is O or S, Y'is -O-, -S- or -NR'-, and a portion.

May be replaced. In some embodiments, Y'is -O- and is -S- or -NH-. In some embodiments

teeth,

Is. In some embodiments

teeth,

Is. In some embodiments

teeth,

Is. In some embodiments, the -X-L-R ¹ is

Has a structure of, where X'is O or S and is a portion.

May be replaced. In some embodiments

teeth,

Is. In some embodiments, the -X-L-R ¹ is

And

May be replaced. In some embodiments, the -X-L-R ¹ is

And

Is replaced. In some embodiments, the -X-L-R ¹ is

And

Is unsubstituted.

から選択される。いくつかの実施形態において、Ｌ^ｘは、

である。いくつかの実施形態において、－Ｘ－Ｌ－Ｒ^１は、（ＣＨ_３）_３Ｃ－Ｓ－Ｓ－Ｌ^ｘ－Ｓ－である。いくつかの実施形態において、－Ｘ－Ｌ－Ｒ^１は、Ｒ^１－Ｃ（＝Ｘ’）－Ｙ’－Ｃ（Ｒ）_２－Ｓ－Ｌ^ｘ－Ｓ－である。いくつかの実施形態において、－Ｘ－Ｌ－Ｒ^１は、Ｒ－Ｃ（＝Ｘ’）－Ｙ’－ＣＨ_２－Ｓ－Ｌ^ｘ－Ｓ－である。いくつかの実施形態において、－Ｘ－Ｌ－Ｒ^１は、

である。 In some embodiments, -X-L-R ¹ is R1 ^- C (O) -S-L ^x -S-, where L ^x is a optionally substituted group.

Is selected from. In some embodiments, L ^x is

Is. In some embodiments, -X-L-R ¹ is (CH ₃ ) _3C -S-S-L ^x -S-. In some embodiments, -X-L-R ¹ is R1 ^- C (= X') -Y'-C (R) ₂ -S-L ^x -S-. In some embodiments, -X-L-R ¹ is RC (= X') -Y'-CH ₂ -S-L ^x -S-. In some embodiments, the -X-L-R ¹ is

Is.

As will be appreciated by those skilled in the art, many of the -X ^- L ^- R units described herein are cleavable and can be converted to -X- after administration to the subject. .. In some embodiments, -XL-R ¹ is cleaveable. In some embodiments, -X-L-R ¹ is -S-L-R ¹ and is converted to ^-S- after administration to the subject. In some embodiments, the enzyme of interest facilitates conversion. As will be appreciated by those skilled in the art, it is known in the art that ^one -S-L-R unit is converted to ^-S- after administration and is used in the study of drug metabolism and pharmacokinetics. It is practiced, including what is done.

である。 In some embodiments, the internucleotide bond having the structure of formula I is

Is.

を有する。
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, the polynucleotide binding of formula I is the structure of formula I-a:

Have.
Here, each variable is independently defined as described above and is as described herein.

を有し、
ここで、各変数は、独立して、上記のとおり定義し、ここに記載したとおりである。 In some embodiments, the internucleotide binding of formula I is the structure of formula Ib:

Have,
Here, each variable is independently defined as described above and is as described herein.

の構造を有するホスホロチオエートトリエステル結合であり、
ここで、Ｐ^＊は、不斉リン原子であり、ＲｐまたはＳｐのいずれかであり；
Ｌは、共有結合または任意に置換され、直鎖または分岐のＣ_１～Ｃ_１０アルキレンであり、ここでＬの１以上のメチレン単位は、置換されていてもよいＣ_１～Ｃ_６アルキレン、Ｃ_１～Ｃ_６アルケニレン、－Ｃ≡Ｃ－、－Ｃ（Ｒ´）_２－、－Ｃｙ－、－Ｏ－、－Ｓ－、－Ｓ－Ｓ－、－Ｎ（Ｒ´）－、－Ｃ（Ｏ）－、－Ｃ（Ｓ）－、－Ｃ（ＮＲ´）－、－Ｃ（Ｏ）Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｃ（Ｏ）Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｃ（Ｏ）－、－Ｎ（Ｒ´）Ｃ（Ｏ）Ｏ－、－ＯＣ（Ｏ）Ｎ（Ｒ´）－、－Ｓ（Ｏ）－、－Ｓ（Ｏ）_２－、－Ｓ（Ｏ）_２Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｓ（Ｏ）_２－、－ＳＣ（Ｏ）－、－Ｃ（Ｏ）Ｓ－、－ＯＣ（Ｏ）－、または－Ｃ（Ｏ）Ｏ－により任意におよび独立して置換され；
Ｒ^１は、ハロゲン、Ｒ、または置換されていてもよいＣ_１～Ｃ_５０脂肪族であり、１以上のメチレン単位は、置換されていてもよいＣ_１～Ｃ_６アルキレン、Ｃ_１～Ｃ_６アルケニレン、－Ｃ≡Ｃ－、－Ｃ（Ｒ´）_２－、－Ｃｙ－、－Ｏ－、－Ｓ－、－Ｓ－Ｓ－、－Ｎ（Ｒ´）－、－Ｃ（Ｏ）－、－Ｃ（Ｓ）－、－Ｃ（ＮＲ´）－、－Ｃ（Ｏ）Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｃ（Ｏ）Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｃ（Ｏ）－、－Ｎ（Ｒ´）Ｃ（Ｏ）Ｏ－、－ＯＣ（Ｏ）Ｎ（Ｒ´）－、－Ｓ（Ｏ）－、－Ｓ（Ｏ）_２－、－Ｓ（Ｏ）_２Ｎ（Ｒ´）－、－Ｎ（Ｒ´）Ｓ（Ｏ）_２－、－ＳＣ（Ｏ）－、－Ｃ（Ｏ）Ｓ－、－ＯＣ（Ｏ）－、または－Ｃ（Ｏ）Ｏ－により任意におよび独立して置換され；
各Ｒ´は、独立して－Ｒ、－Ｃ（Ｏ）Ｒ、－ＣＯ_２Ｒ、もしくは－ＳＯ_２Ｒであるか、または：
同じ窒素上の２つのＲ´は、それらの介在原子と一緒になって置換されていてもよいヘテロ環式、もしくはヘテロアリール環を形成するか、または
同じ炭素上の２つのＲ´は、それらの介在原子と一緒になって、置換されていてもよいアリール、炭素環式、ヘテロ環式、またはヘテロアリール環を形成し；
－Ｃｙ－は、フェニレン、カルボシクリレン、アリーレン、ヘテロアリーレン、またはヘテロシクリレンから選択される置換されていてもよい２価の環であり；
各Ｒは、独立して水素であるか、または、Ｃ_１～Ｃ_６脂肪族、フェニル、カルボシクリル、アリール、ヘテロアリール、またはヘテロシクリルから選択される置換されていてもよい基であり；および
各

は、独立してヌクレオシドとの結合を表し；
Ｒ^１は、Ｌが共有結合のとき、－Ｈ以外である。 In some embodiments, the internucleotide binding of formula I is such that formula Ic:

It is a phosphorothioate triester bond having the structure of
Where P ^* is an asymmetric phosphorus atom, either Rp or Sp;
L is a covalently bonded or optionally substituted, linear or branched C ₁ to C ₁₀ alkylene, wherein one or more methylene units of L may be substituted C ₁ to C ₆ alkylene, C. ₁ to C ₆ alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R')-, -C ( O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-, -S (O) _2- , -S (O) ₂ N (R')-,-N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O)-, or- Arbitrarily and independently replaced by C (O) O-;
R ¹ is a halogen, R, or optionally substituted C ₁ to C ₅₀ aliphatic, and one or more methylene units may be substituted C ₁ to C ₆ alkylene, C ₁ to C ₆ Alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R')-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R')-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-, -S (O) _2- , -S (O) ) ₂ N (R')-,-N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O)-, or -C (O) Replaced arbitrarily and independently by O-;
Each R'is independently -R, -C (O) R, -CO ₂ R, or -SO ₂ R, or:
Two R'on the same nitrogen form a heterocyclic or heteroaryl ring that may be substituted together with their intervening atoms, or two R'on the same carbon are them. Together with the intervening atoms of, it forms an aryl, carbocyclic, heterocyclic, or heteroaryl ring that may be substituted;
-Cy- is a optionally substituted divalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene;
Each R is an independently hydrogen or is a _{optionally substituted group selected from C 1-6 aliphatic} , phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl; and each

Represents the binding to the nucleoside independently;
R ¹ is other than −H when L is a covalent bond.

である。 In some embodiments, the internucleotide bond having the structure of formula I is

Is.

である。 In some embodiments, the internucleotide bonds having the structure of formula Ic are

Is.

In some embodiments, the invention is chiral controlled comprising one or more phosphate diester bonds and one or more modified oligonucleotide bonds having the formulas Ia, Ib, or Ic. Provide oligonucleotides.

In some embodiments, the invention provides a chiral-controlled oligonucleotide comprising at least one phosphate diester internucleotide bond and at least one phosphodiester triester bond having the structure of formula Ic. .. In some embodiments, the invention provides a chiral-controlled oligonucleotide comprising at least one phosphate diester polynucleotide bond and at least two phosphodiester triester bonds having the structure of formula Ic. .. In some embodiments, the invention provides a chiral-controlled oligonucleotide comprising at least one phosphate diester polynucleotide bond and at least three phosphorothioate triester bonds having the structure of formula Ic. .. In some embodiments, the invention provides a chiral-controlled oligonucleotide comprising at least one phosphate diester polynucleotide bond and at least four phosphorothioate triester bonds having the structure of formula IC. .. In some embodiments, the invention provides a chiral-controlled oligonucleotide comprising at least one phosphate diester internucleotide bond and at least five phosphorothioate triester bonds having the structure of formula Ic. ..

In some embodiments, the invention provides chirally controlled oligonucleotides comprising the sequences shown in any of the annexes of the present application. In some embodiments, the invention provides chirally controlled oligonucleotides comprising the sequences shown in Annex A. In some embodiments, the invention provides chirally controlled oligonucleotides comprising the sequences shown in Annex B. In some embodiments, the invention provides chirally controlled oligonucleotides comprising the sequences shown in Annex C. In some embodiments, the invention provides a chirally controlled oligonucleotide having the sequence shown in any of the annexes of the present application. In some embodiments, the invention provides a chirally controlled oligonucleotide having the sequences shown in Annex A. In some embodiments, the invention provides a chirally controlled oligonucleotide having the sequences shown in Annex B. In some embodiments, the invention provides a chirally controlled oligonucleotide having the sequence shown in Annex C.

In some embodiments, the present invention provides chirally controlled oligonucleotides comprising the sequences set forth in GCCTCAGTCTGCTTCGCACC. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence set forth in GCCTCAGTCTGCTTCGCACC, wherein the sequence has more than 50% identity with GCTCAGTCTGCTTCGCACC. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence set forth in GCCTCAGTCTGCTTCGCACC, wherein the sequence has more than 60% identity with GCTCAGTCTGCTTCGCACC. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence set forth in GCCTCAGTCTGCTTCGCACC, wherein the sequence has 70% or more identity with GCTCAGTCTGCTTCGCACC. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence shown in GCCTCAGTCTGCTTCGCACC, which sequence has more than 80% identity with GCCTCAGTCTGCTTCGCACC. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence set forth in GCCTCAGTCTGCTTCGCACC, wherein the sequence has 90% or more identity with GCTCAGTCTGCTTCGCACC. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence set forth in GCCTCAGTCTGCTTCGCACC, wherein the sequence has 95% or more identity with GCTCAGTCTGCTTCGCACC. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC. In some embodiments, the invention provides a chirally controlled oligonucleotide having a sequence of GCCTCAGTCTGCTTCGCACC.

である。いくつかの実施形態において、本発明は、ＧＣＣＴＣＡＧＴＣＴＧＣＴＴＣＧＣＡＣＣに示される配列を含むキラル制御されたオリゴヌクレオチドを提供し、
各インターヌクレオチド結合は、

である。いくつかの実施形態において、本発明は、ＧＣＣＴＣＡＧＴＣＴＧＣＴＴＣＧＣＡＣＣに示される配列を含むキラル制御されたオリゴヌクレオチドを提供し、少なくとも１個のインターヌクレオチド結合は、

である。いくつかの実施形態において、本発明は、ＧＣＣＴＣＡＧＴＣＴＧＣＴＴＣＧＣＡＣＣに示される配列を含むキラル制御されたオリゴヌクレオチドを提供し、各インターヌクレオチド結合は、

である。 In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence set forth in GCCTCAGTCTGCTTCGCACC, at least one internucleotide binding having a chirally bound phosphate. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence set forth in GCCTCAGTCTGCTTCGCACC, at least one internucleotide binding having the structure of formula I. In some embodiments, the present invention provides chirally controlled oligonucleotides comprising the sequences set forth in GCCTCAGTCTGCTTCGCACC, each internucleotide binding having the structure of formula I. In some embodiments, the invention provides a chirally controlled oligonucleotide containing the sequence set forth in GCCTCAGTCTGCTTCGCACC, at least one internucleotide binding having the structure of formula Ic. In some embodiments, the present invention provides chirally controlled oligonucleotides comprising the sequences set forth in GCCTCAGTCTGCTTCGCACC, where each internucleotide binding has a structure of formula Ic. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence set forth in GCCTCAGTCTGCTTCGCACC, wherein at least one internucleotide binding is present.

Is. In some embodiments, the present invention provides chirally controlled oligonucleotides comprising the sequences set forth in GCCTCAGTCTGCTTCGCACC.
Each nucleotide bond is

Is. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence set forth in GCCTCAGTCTGCTTCGCACC, wherein at least one internucleotide binding is present.

Is. In some embodiments, the present invention provides chirally controlled oligonucleotides comprising the sequences set forth in GCCTCAGTCTGCTTCGCACC, where each internucleotide binding is:

Is.

である。本発明は、ＧＣＣＴＣＡＧＴＣＴＧＣＴＴＣＧＣＡＣＣの配列を含むキラル制御されたオリゴヌクレオチド提供し、各インターヌクレオチド結合は、

である。本発明は、ＧＣＣＴＣＡＧＴＣＴＧＣＴＴＣＧＣＡＣＣの配列を含むキラル制御されたオリゴヌクレオチド提供し、少なくとも１個のインターヌクレオチド結合は、

である。本発明は、ＧＣＣＴＣＡＧＴＣＴＧＣＴＴＣＧＣＡＣＣの配列を含むを」キラル制御されたオリゴヌクレオチド提供し、各インターヌクレオチド結合は、

である。 In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC, at least one internucleotide binding having a chirally bound phosphate. The present invention provides a chirally controlled oligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC, at least one internucleotide binding having the structure of formula I. The present invention provides chirally controlled oligonucleotides comprising the sequence of GCCTCAGTCTGCTTCGCACC, each internucleotide binding having the structure of formula I. The present invention provides a chirally controlled oligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC, at least one internucleotide binding having the structure of formula Ic. The present invention provides chirally controlled oligonucleotides comprising the sequence of GCCTCAGTCTGCTTCGCACC, each internucleotide binding having a structure of formula Ic. The present invention provides chirally controlled oligonucleotides comprising the sequence of GCCTCAGTCTGCTTCGCACC, wherein at least one polynucleotide binding is.

Is. The present invention provides chirally controlled oligonucleotides comprising the sequence of GCCTCAGTCTGCTTCGCACC, where each internucleotide binding is:

Is. The present invention provides chirally controlled oligonucleotides comprising the sequence of GCCTCAGTCTGCTTCGCACC, wherein at least one polynucleotide binding is.

Is. The present invention provides chirally controlled oligonucleotides that include the sequence of GCCTCAGTCTGCTTCGCACC, where each internucleotide binding is:

Is.

である。いくつかの実施形態において、本発明は、ＧＣＣＴＣＡＧＴＣＴＧＣＴＴＣＧＣＡＣＣの配列を含むキラル制御されたオリゴヌクレオチドを提供し、各インターヌクレオチド結合は、

である。いくつかの実施形態において、本発明は、ＧＣＣＴＣＡＧＴＣＴＧＣＴＴＣＧＣＡＣＣの配列を含むキラル制御されたオリゴヌクレオチドを提供し、少なくとも１個のインターヌクレオチド結合は、

である。いくつかの実施形態において、本発明は、ＧＣＣＴＣＡＧＴＣＴＧＣＴＴＣＧＣＡＣＣの配列を含むキラル制御されたオリゴヌクレオチドを提供し、各インターヌクレオチド結合は、

である。 In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC, at least one internucleotide binding having a chirally bound phosphate. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC, at least one internucleotide binding having the structure of formula I. In some embodiments, the invention provides a chirally controlled oligonucleotide containing the sequence of GCCTCAGTCTGCTTCGCACC, where each internucleotide binding has the structure of formula I. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC, at least one internucleotide binding having the structure of formula Ic. In some embodiments, the invention provides a chirally controlled oligonucleotide containing the sequence of GCCTCAGTCTGCTTCGCACC, where each internucleotide binding has a structure of formula Ic. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC, wherein at least one polynucleotide binding is present.

Is. In some embodiments, the present invention provides chirally controlled oligonucleotides comprising the sequence of GCCTCAGTCTGCTTCGCACC, where each internucleotide binding is:

Is. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC, wherein at least one polynucleotide binding is present.

Is. In some embodiments, the present invention provides chirally controlled oligonucleotides comprising the sequence of GCCTCAGTCTGCTTCGCACC, where each internucleotide binding is:

Is.

In some embodiments, the invention provides a chirally controlled oligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC, at least one bound phosphate is Rp. It will be appreciated by those skilled in the art that, in certain embodiments, the chirally controlled oligonucleotide comprises an RNA sequence, where each T is independently and optionally replaced with U. In some embodiments, the invention provides a chirally controlled oligonucleotide containing the sequence of GCCTCAGTCTGCTTCGCACC, each of the bound phosphates being Rp. In some embodiments, the invention provides a chirally controlled oligonucleotide containing the sequence of GCCTCAGTCTGCTTCGCACC, at least one bound phosphate is Sp. In some embodiments, the invention provides a chirally controlled oligonucleotide containing the sequence of GCCTCAGTCTGCTTCGCACC, each of the bound phosphates being Sp. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising a sequence of GCCTCAGTCTGCTTCGCACC, wherein the oligonucleotide is blockmer. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising a sequence of GCCTCAGTCTGCTTCGCACC, wherein the oligonucleotide is a stereoblockmer. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising a sequence of GCCTCAGTCTGCTTCGCACC, wherein the oligonucleotide is a P-modified blocker. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising a sequence of GCCTCAGTCTGCTTCGCACC, wherein the oligonucleotide is a binding blocker. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising a sequence of GCCTCAGTCTGCTTCGCACC, wherein the oligonucleotide is an altomer. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising a sequence of GCCTCAGTCTGCTTCGCACC, wherein the oligonucleotide is a stereoaltomer. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising a sequence of GCCTCAGTCTGCTTCGCACC, wherein the oligonucleotide is a P-modified altomer. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising a sequence of GCCTCAGTCTGCTTCGCACC, wherein the oligonucleotide is a bound altomer. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising a sequence of GCCTCAGTCTGCTTCGCACC, wherein the oligonucleotide is a unimmer. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising a sequence of GCCTCAGTCTGCTTCGCACC, wherein the oligonucleotide is a stereo-unimer. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising a sequence of GCCTCAGTCTGCTTCGCACC, wherein the oligonucleotide is a P-modified unimmer. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising a sequence of GCCTCAGTCTGCTTCGCACC, wherein the oligonucleotide is a binding unimmer. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising a sequence of GCCTCAGTCTGCTTCGCACC, wherein the oligonucleotide is a gapmer. In some embodiments, the invention provides a chirally controlled oligonucleotide comprising a sequence of GCCTCAGTCTGCTTCGCACC, wherein the oligonucleotide is Skipmer.

In some embodiments, the invention provides a chiral-controlled oligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC, where each cytosine is optionally and independently substituted with 5-methylcytosine. In some embodiments, the invention provides a chiral-controlled oligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC, where at least one cytosine is optionally and independently substituted with 5-methylcytosine. In some embodiments, the invention provides a chiral-controlled oligonucleotide comprising the sequence of GCCTCAGTCTGCTTCGCACC, where each cytosine is optionally and independently substituted with 5-methylcytosine. Exemplary chirally controlled oligonucleotides containing the sequence of GCCTCAGTCTGCTTCGCACC are shown in Table 2 below.

Exemplary chirally controlled oligonucleotides

In some embodiments, chirally controlled oligonucleotides are designed such that one or more nucleotides contain a phosphate modification that facilitates auto-release under certain circumstances. That is, under certain conditions, certain phosphorus modifications are designed to self-cleave from oligonucleotides to obtain, for example, phosphodiesters found in native DNA and RNA. In some embodiments, such phosphoric acid modifications have a structure of —OL—R ¹ , each of L and R ¹ is independently defined and herein as described above. As described. In some embodiments, the autorelease group comprises a morpholino group. In some embodiments, the autorelease group is characterized by the ability to deliver the agent to the phosphate linker of the polynucleotide, which facilitates the modification of the phosphorus atom, for example by desulfurization. In some embodiments, the agent is water, which is further modified by hydrolysis to form the phosphodiester found in natural DNA and RNA.

In some embodiments, chirally controlled oligonucleotides are designed so that the properties of the resulting agent are improved through one or more specific modifications with phosphoric acid. The rapid degradation of certain oligonucleotides by nucleases and reduced uptake of cells through the cell membrane has been described in many documents in the art (Poijarvi-Virta et al., Curr. Med. Chem. (2006), 13 (28); 3441-65; Wagner et al., Med. Res. Rev. (2000), 20 (6): 417-51; Peyrottes et al., Mini Rev. Med. Chem. ( 2004), 4 (4): 395-408; Gosselin et al., (1996), 43 (1): 196-208; Bologna et al., (2002), Nuclease & Nucleic Acid Drug Develoment 12: 33. ). For example, Vives et al. (Nucleic Acids Research (1999), 27 (20): 4071-76) showed that the cell permeability of tert-butyl SATE pro-oligonucleotides was significantly increased when compared to the parent oligonucleotide. I found that. ..

In some embodiments, modifications in bound phosphate are characterized by the ability to be converted to phosphate diesters such as those present in native DNA and RNA, with one or more esterases, nucleases, and / or cytochrome P450. It is converted by, but is not limited to, enzymes, those listed in Table 3 below.
Illustrative enzyme

In some embodiments, the modification in phosphate results in a P-modified moiety characterized by functioning as a prodrug, eg, the P-modified moiety is delivered to the desired position prior to removal of the oligonucleotide. Make it easier. For example, in some embodiments, the P-modified moiety is due to pegging with bound phosphate. Those skilled in the art of related art will appreciate that different PEG chain lengths are useful and the choice of chain length is partly determined by the outcome to be achieved by PEGylation. .. For example, in some embodiments, PEGylation has the effect of reducing RES uptake and prolonging the in vivo circulating life of oligonucleotides.

In some embodiments, the reagents used for PEGylation according to the invention have a molecular weight of about 300 g / mol to about 100,000 g / mol. In some embodiments, the molecular weight of the reagent used for PEGylation is from about 300 g / mol to about 10,000 g / mol. In some embodiments, the molecular weight of the reagent used for PEGylation is from about 300 g / mol to about 5,000 g / mol. In some embodiments, the molecular weight of the reagent used for PEGylation is about 500 g / mol. In some embodiments, the molecular weight of the reagent used for PEGylation is about 1000 g / mol. In some embodiments, the molecular weight of the reagent used for PEGylation is about 3000 g / mol. In some embodiments, the molecular weight of the reagent used for PEGylation is about 5000 g / mol.

In certain embodiments, the reagent used for PEGylation is PEG500. In certain embodiments, the reagent used for PEGylation is PEG1000. In certain embodiments, the reagent used for PEGylation is PEG3000. In certain embodiments, the reagent used for PEGylation is PEG5000.

In some embodiments, the P-modified moiety is characterized by functioning as a PK enhancer for, for example, lipids, PEGylated lipids and the like.

In some embodiments, the P-modified moiety is characterized by functioning as an agent that promotes cell entry and / or endosome avoidance, such as membrane-destroying lipids or peptides.

In some embodiments, the P-modified moiety is characterized by functioning as a target agent. In some embodiments, the P-modified moiety is or comprises a target agent. The term "targeted agent" herein is associated with the payload of interest (eg, in association with an oligonucleotide or oligonucleotide composition) and interacts with the target site of interest. Therefore, the target payload targets the target site when it is associated with substantially more target drug than it is confirmed or under equivalent circumstances when the target payload is not associated with the target drug. To. The target agent may be, or includes, for example, any of a variety of chemical moieties such as small molecule moieties, nucleic acids, polypeptides, carbohydrates and the like. Target agents are further described in Adarsh et al. , "Organelle Special Targeted Drug Delivery-A Review," International Journal of Research in Pharmaceutical and Biomedical Sciences. 895.

Exemplary such targeting agents include, but are not limited to, proteins (eg, transferase), oligopeptides (eg, cyclic and acyclic RGD-containing oligopeptides), antibodies (monoclonal antibodies and poly). Cloning antibodies such as IgG, IgA, IgM, IgD, IgE antibodies), sugars / carbohydrates (eg monosaccharides and / or oligosaccharides (mannose, mannose-6-phosphate, galactose, etc.)), vitamins (eg, galactose, etc.) Folic acid), or another small biomolecule. In some embodiments, the target moiety is a steroid molecule (eg, bile acids including cholic acid, deoxycholic acid, dehydrocholic acid; cortisone; digoxygenin; testosterone; cholesterol; dual at the 3-position of the cortisone ring. Cationic steroids such as cortisone with a trimethylaminomethylhydrazide group that binds through binding). In some embodiments, the target moiety is a lipophilic molecule (eg, alicyclic hydrocarbons, saturated and unsaturated fatty acids, waxes, terpenes, and polyalicyclic hydrocarbons such as enamel and buckminster fullerene). be. In some embodiments, the fat-soluble molecule is a terpenoid such as vitamin A, retinoic acid, retinal, or dehydroretinal. In some embodiments, the target moiety is a peptide.

In some embodiments, the P-modified moiety is the target agent of formula --X-L-R ¹ , each of X, L, and R ¹ as defined in Formula I above. be.

In some embodiments, the P-modified moiety is characterized by facilitating specific delivery of cells.

In some embodiments, the P-modified moiety is characterized by being included in one or more of the categories described above. For example, in some embodiments, the P-modified moiety functions as a PK enhancer and target ligand. In some embodiments, the P-modified moiety functions as a prodrug and an endosome avoidant. Those skilled in the art of the art will appreciate that many other such combinations are possible and contemplated by the present invention.

Nucleobases In some embodiments, the nucleobases present in the provided oligonucleotides are natural nucleobases or modified nucleobases derived from natural nucleobases. Examples include, but are not limited to, uracil, thymine, adenine, cytosine, and guanine, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5 in which each amino group is protected by an acyl protective group. -Pyrimidine analogs such as iodouracil, 2,6-diaminopurine, azacitosine, pseudo-isocytosine and pseudo-uracil, and other modified nucleobases such as 8-substituted purine, xanthin, or hypoxanthin (last). Two are spontaneous decomposition products). Exemplary modified nucleobases are Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196 and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. . Disclosures in 7, 313.

ここで、Ｒ^８は、置換されていてもよい、脂肪族、アリール、アラルキル、アリールオキシルアルキル、カルボシクリル、１～１５個の炭素原子を有するヘテロシクリルまたはヘテロアリール基から選択される直鎖または分岐の基であり、例にすぎないが、メチル、イソプロピル、フェニル、ベンジル、またはフェノキシメチル基が含まれ；およびＲ^９ならびにＲ^１０のそれぞれは、独立して直鎖または分岐の脂肪族、カルボシクリル、アリール、ヘテロシクリルおよびヘテロアリールから選択される置換されていてもよい基である。 Compounds represented by the following general formula are also contemplated as modified nucleobases:

Here, R ⁸ is a straight chain or branched selected from an aliphatic, aryl, aralkyl, aryloxylalkyl, carbocyclyl, heterocyclyl having 1 to 15 carbon atoms or a heteroaryl group which may be substituted. Groups, and only examples, include methyl, isopropyl, phenyl, benzyl, or phenoxymethyl groups; and each of R ⁹ and R ¹⁰ is an independently linear or branched aliphatic, carbocyclyl, aryl. , A optionally substituted group selected from heterocyclyls and heteroaryls.

Modified nucleobases also include expanded sized nucleobases to which one or more aryl rings, such as phenyl rings, have been added. Substitution of nucleobases is available at the Glen Research catalog (www.glenresearch.com); Krueger AT et al, Acc. Chem. Res., 2007, 40, 141-150; Kool, ET, Acc. Chem. Res., 2002 , 35, 936-943; Benner SA, et al., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, FE, et al., Curr. Opin. Chem. Biol., 2003, 7, 723-733; Hirao, I., Curr. Opin. Chem. Biol., 2006,10,622-627 and is believed to be useful in the synthesis of the nucleic acids described herein. Some examples of nucleobases of expanded size are as follows.

Here, the modified nucleobase also contains a structure that is not considered to be a nucleobase, but is, for example, another part, such as, but not limited to, a choline or porphyrin-induced ring. Porphyrin-induced base substitutions are described in Morales-Rojas, H and Kool, ET, Org. Lett., 2002, 4, 4377-4380. An example of a porphyrin-induced ring used as a base substitution is shown below:

。 In some embodiments, the modified nucleobase has one of the following structures and may be substituted:

..

。 In some embodiments, the modified nucleobase is fluorescent. Illustrative modified nucleobases of such fluorescence include phenanthrene, pyrene, stilbene, isoxanthine, isozanthopterin, terphenyl, terthiophene, benzoterthiophene, coumarin, rumazine, as shown below. Includes tether stilbene, benzo-uracil, and naphtho-uracil:

..

In some embodiments, the modified nucleobase is unsubstituted. In some embodiments, the modified nucleobase is substituted. In some embodiments, the modified nucleobase is substituted to include, for example, a heteroatom, an alkyl group, or a binding moiety attached to a fluorescent moiety, a biotin or avidin moiety, or another protein or peptide. .. In some embodiments, the modified nucleobase is not a nucleobase in the most classical sense, but a "universal base" that functions like a nucleobase. A typical example of such a universal base is 3-nitropyrrole.

In some embodiments, another nucleoside can also be used in the processes disclosed herein to incorporate a modified nucleobase or a nucleobase that covalently binds to a modified sugar. include. Some examples of nucleosides incorporating modified nucleic acid bases include 4-acetylcytidine; 5- (carboxyhydroxylmethyl) uridine; 2'-O-methylcytidine;5-carboxymethylaminomethyl-2-thiouridine; 5 -Carboxymethylaminomethyluridine; Dihydrouridine; 2'-O-methylpsoiduridine; Beta D-galactosylqueosine; 2'-O-methylguanosine; N6 ^- isopentenyladenosine; 1 -Methyluridine; 1-methyluridine; 1-methylguanosine; l-methylinosin; 2,2-dimethylguanosine; 2-methyladenosine; 2-methylguanosine; N7 ^- methylguanosine; 3-methyl-cytidine; 5-Methylcytidine; 5-Hydroxymethylcytidine; 5-formylcytosine; 5-carboxylcytosine; N6-methylcytidine; 7-methylguanosine; ⁵ -methylaminoethyluridine; 5-methoxyaminomethyl-2-thiouridine; beta D-mannosylqueosine; 5-methoxycarbonylmethyluridine; 5-methoxyuridine; 2-methylthio-N6-isopentenyladenosine; N-(( ⁹ -beta D ribofuranosyl-2-methylthiopurine) -6-yl) carbamoyl) threonine; N-((9-betaD ribofuranosylpurine-6-yl) -N-methylcarbamoyl) treonin; uridine-5-oxyacetic acid methyl ester; uridine-5-oxyacetic acid ( v); pseudouridine; queosine; 2-thiocytidine; 5-methyl-2-thiouridine; 2-thiouridine; 4-thiouridine; 5-methyluridine; 2'-O-methyl-5-methyluridine; and 2' -O-methyluridine.

In some embodiments, the nucleoside comprises a 6'-modified bicyclic nucleoside analog having the chirality of either (R) or (S) at the 6'position, US Pat. No. 7,399,845. Includes analogs described in the issue. In another embodiment, the nucleoside comprises a 5'-modified bicyclic nucleoside analog having the chirality of either (R) or (S) at the 5'position and is described in US Patent Application Publication No. 20070287831. Includes analogs that are.

In some embodiments, the nucleobase or modified nucleobase comprises one or more biomolecular binding moieties such as, for example, antibodies, antibody fragments, biotin, avidin, streptavidin, receptor ligands, or chelating moieties. In another embodiment, the nucleobase or modified nucleobase is 5-bromouracil, 5-iodouracil, or 2,6-diaminopurine. In some embodiments, the nucleobase or modified nucleobase is modified by fluorescence or replacement of a biomolecular binding moiety. In some embodiments, the substituent on the nucleobase or modified nucleobase is the fluorescent moiety. In some embodiments, the substituent or modified nucleobase on the nucleobase is biotin or avidin.

Representative US patents that teach the fabrication of certain of the above modified nucleobases and other modified nucleobases are, but are not limited to, the above-mentioned US Pat. No. 3,678,808. And US Pat. No. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432, 272; 5,457,187; 5,457,191; 5,459,255; 5,484,908; 5,502,177; 5,525,711 No. 5,552,540; No. 5,587,469; No. 5,594,121, No. 5,596,091; No. 5,614,617; No. 5,681,941; No. 5, 750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380, 368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088. , And these are incorporated herein by reference.

Sugars The most common natural nucleotides consist of nucleobases, adenosine (A), cytosine (C), guanine (G), and ribose sugars bound to thymine (T) or uracil (U). Also contemplated are modified nucleotides in which the phosphate group or bound phosphate within the nucleotide is capable of binding to various positions in the sugar or modified sugar. As a non-limiting example, a phosphate group or bound phosphate can be attached to the 2', 3', 4'or 5'hydroxyl moieties of a sugar or modified sugar. Nucleotides incorporating the modified nucleobases described herein are also contemplated in this context. In some embodiments, nucleotides containing unprotected-OH moieties or modified nucleotides are used by the methods of the invention.

Another modified sugar is also incorporated within the provided oligonucleotide. In some embodiments, the modified sugar is one of the following: -F; -CF ₃ , -CN, -N ₃ , -NO, -NO ₂ , -OR', -SR', or. -N (R') contains one or more substituents containing ₂ at the 2-position, where each R'is independently defined as described above and as described herein; -O- (C). ₁ to C ₁₀ alkyl), -S- (C ₁ to C ₁₀ alkyl), -NH- (C ₁ to C ₁₀ alkyl), or -N (C ₁ to C ₁₀ alkyl) ₂ ; -O- (C ₂ ) -C ₁₀ alkenyl), -S- (C ₂ -C ₁₀ alkenyl), -NH- (C ₂ -C ₁₀ alkenyl), or -N (C ₂ -C ₁₀ alkenyl) ₂ ; -O- (C _2-- C ₁₀ alkynyl), -S- (C ₂ -C ₁₀ alkynyl), -NH- (C ₂ -C ₁₀ alkynyl), or -N (C ₂ -C ₁₀ alkynyl) ₂ ; or -O- (C ₁ ) -C ₁₀ alkylene) -O- (C ₁ to C ₁₀ alkyl), -O- (C ₁ to C ₁₀ alkylene) -NH- (C ₁ to C ₁₀ alkyl) or -O- (C ₁ to C ₁₀ ) Alkylene) -NH (C ₁ to C ₁₀ alkyl) ₂ , -NH- (C ₁ to C ₁₀ alkylene) -O- (C ₁ to C ₁₀ alkyl), or -N (C ₁ to C ₁₀ alkyl)-( C ₁ to C ₁₀ alkylene) -O- (C ₁ to C ₁₀ alkyl), where the alkyl, alkylene, alkenyl and alkynyl may be substituted or unsubstituted. Examples of substituents include, but are not limited to, —O (CH ₂ ) _n OCH ₃ and —O (CH ₂ ) _n NH ₂ , where n is from 1 to about 10. Examples include MOE, DMAOE and DMAEOE. Also contemplated herein are the modified sugars described in International Publication No. 2001/088198; and Martin et al., Helv. Chim. Acta, 1995, 78, 486-504. In some embodiments, the modified sugar is a substituted silyl group, RNA cleavage group, reporter group, fluorescent label, intercalator, group that enhances the pharmacokinetics of the nucleic acid, and improves the pharmacological properties of the nucleic acid. Contains one or more groups selected from the groups to cause or another substituent having similar properties. In some embodiments, the 2', 3', 4', 5', of a sugar or modified sugar comprising the 3'position of the sugar of the 3'-terminal nucleotide or the 5'position of the 5'-terminal nucleotide, Alternatively, modification is performed with 1 or more of the 6'position.

In some embodiments, the ribose 2'-OH is
Below: -H, -F; with a substituent containing one of -CF ₃ , -CN, -N ₃ , -NO, -NO ₂ , -OR', -SR', or -N (R') ₂ . Substituted, where each R'is independently defined as described above and as described herein; -O- (C ₁ to C ₁₀ alkyl), -S- (C ₁ to C ₁₀ alkyl). ), -NH- (C ₁ to C ₁₀ alkyl), or -N (C ₁ to C ₁₀ alkyl) ₂ ; -O- (C ₂ -C ₁₀ alkenyl), -S- (C ₂ -C ₁₀ alkenyl) , -NH- (C ₂ -C ₁₀ alkenyl), or -N (C ₂ -C ₁₀ alkenyl) ₂ ; -O- (C ₂ -C ₁₀ alkynyl), -S- (C ₂ -C ₁₀ alkynyl), -NH- (C ₂ -C ₁₀ alkynyl) or -N (C ₂ -C ₁₀ alkynyl) ₂ ; or -O-- (C ₁ to C ₁₀ alkylene) -O-- (C ₁ to C ₁₀ alkyl) , -O- (C ₁ to C ₁₀ alkylene) -NH- (C ₁ to C ₁₀ alkyl) or -O- (C ₁ to C ₁₀ alkylene) -NH (C ₁ to C ₁₀ alkyl) ₂ , -NH- (C ₁ to C ₁₀ alkylene) -O- (C ₁ to C ₁₀ alkyl) or -N (C ₁ to C ₁₀ alkyl)-(C ₁ to C ₁₀ alkylene) -O- (C ₁ to C ₁₀ alkyl) ), Where alkyl, alkylene, alkenyl and alkynyl may be substituted or unsubstituted. In some embodiments, 2'-OH is replaced by -H (deoxyribose). In some embodiments, 2'-OH is replaced by -F. In some embodiments, 2'-OH is replaced by -OR'. In some embodiments, 2'-OH is replaced by -OMe. In some embodiments, 2'-OH is replaced by -OCH ₂ CH ₂ OMe.

The modified sugar also contains a locked nucleic acid (LNA). In some embodiments, the locked nucleic acid has the structure shown below. In a locked nucleic acid having the following structure, Ba represents a nucleobase or a modified nucleobase as described herein, and R ^2s is -OCH ₂ C4'-.

In some embodiments, the modified sugar is, for example, ENA as described in Sethet al., Jam Chem Soc. 2010 October 27; 132 (42): 14942-14950. In some embodiments, the modified sugar is one found in XNAs (xenonucleic acids), such as arabinose, anhydrohexitol, threose, 2'fluoroarabinose, or cyclohexene.

Modified sugars contain imitations of sugars such as cyclobutyl or cyclopentyl moieties instead of pentoflanosyl sugars. Representative US patents that teach the preparation of such modified sugar structures are, but are not limited to, US Pat. Nos. 4,981,957; 5,118,800; 5,319,080. Nos.; and Nos. 5,359,044. Some of the engineered modified sugars include those in which the oxygen atom of the ribose ring is replaced with nitrogen, sulfur, selenium, or carbon. In some embodiments, the modified sugar is modified ribose, the oxygen atom in the ribose ring is replaced with nitrogen, and the nitrogen is replaced with an alkyl group (eg, methyl, ethyl, isopropyl, etc.). It may have been.

。 Non-limiting examples of modified sugars include glycerol forming glycerol nucleic acid (GNA) analogs. An example of a GNA analog is shown below, Zhang, R et al., J. Am. Chem. Soc., 2008, 130, 5846-5847; Zhang L, et al., J. Am. Chem. Soc. ., 2005, 127, 4174-4175 and Tsai CH et al., PNAS, 2007, 14598-14603 (X = ^O- ):

..

。 Another example of a GNA-induced analog, a flexible nucleic acid (FNA) based on a mixed acetal aminal of formylglycerol, is Joyce GF et al., PNAS, 1987, 84, 4398-4402 and Heuberger BD and Switzer C, It is described in J. Am. Chem. Soc., 2008, 130, 412-413 and is as follows:

..

，
のいずれか１つであり、
ここで、Ｘ^ｓは、本明細書中に記載されるＰ－修飾基「－ＸＬＲ^１」に対応し、Ｂａは、ここに定義するとおりである。 Further non-limiting examples of modified sugars are hexopyranosyl (6'-4'), pentopyranosyl (4'-2'), pentopyranosyl (4'-3'), or tetrofuranosyl (3'-2') sugars. Can be mentioned. In some embodiments, the hexopyranosyl (6'-4') sugar has the following formula:

， ，
Is one of
Here, X ^s corresponds to the P-modifying group "-XLR ¹ " described herein, and Ba is as defined herein.

のいずれか１つであり、
ここで、Ｘ^ｓは、本明細書中に記載されるＰ－修飾基「－ＸＬＲ^１」に対応し、Ｂａは、ここに定義するとおりである。 In some embodiments, the pentopyranosyl (4'-2') sugar has the following formula:

Is one of
Here, X ^s corresponds to the P-modifying group "-XLR ¹ " described herein, and Ba is as defined herein.

，
のいずれか１つであり、
ここで、Ｘ^ｓは、本明細書中に記載されるＰ－修飾基「－ＸＬＲ^１」に対応し、Ｂａは、ここに定義するとおりである。 In some embodiments, the pentopyranosyl (4'-3') sugar has the following formula:

， ，
Is one of
Here, X ^s corresponds to the P-modifying group "-XLR ¹ " described herein, and Ba is as defined herein.

,
のいずれかであり、
ここで、Ｘ^ｓは、本明細書中に記載されるＰ－修飾基「－ＸＬＲ^１」に対応し、Ｂａは、ここに定義するとおりである。 In some embodiments, the tetrofuranosyl (3'-2') sugar has the following formula:

,,
Is one of
Here, X ^s corresponds to the P-modifying group "-XLR ¹ " described herein, and Ba is as defined herein.

のいずれか１つであり、
ここで、Ｘ^ｓは、本明細書中に記載されるＰ－修飾基「－ＸＬＲ^１」に対応し、Ｂａは、ここに定義するとおりである。 In some embodiments, the modified sugar has the following formula:

Is one of
Here, X ^s corresponds to the P-modifying group "-XLR ¹ " described herein, and Ba is as defined herein.

In some embodiments, one or more hydroxyl groups in the sugar moiety may be independently substituted with halogen, R'-N (R') ₂ , -OR', or -SR', each R. 'Independently defined as above and as described here.

In some embodiments, the sugar mimics are as shown below, where Xs corresponds to the P-modifying group " ^{-XLR 1} " described herein and Ba is here. X ¹ is selected from -S-, -Se-, -CH _2- , -NMe-, -NET- or -NiPr-.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31% , 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48. %, 49%, 50%, or more (eg, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) (including them) The sugar of the chirally controlled oligonucleotide composition is modified. In some embodiments, only the purine residue is modified (eg, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11). %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44% , 45%, 46%, 47%, 48%, 49%, 50%, or more [eg 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 % Or more] purine residue is modified). In some embodiments, only pyrimidine residues are modified (eg, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11). %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44% , 45%, 46%, 47%, 48%, 49%, 50%, or more [eg 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95. % Or more] pyridimine residues are modified). In some embodiments, both purine and pyrimidine residues are modified.

Modified sugars and sugar mimetics can be prepared by known methods and, without limitation, A. Eschenmoser, Science (1999), 284: 2118; M. Bohringeretal, Helv. Chim. Acta. (1992), 75: 1416-1477; M.Eglietal, J.Am.Chem.Soc. (2006), 128 (33): 10847-56; A.Eschenmoserin Chemical Synthesis: GnosistoPrognosis, C.ChatgilialogluandV.Sniekus, Ed ., (Kluwer Academic, Netherlands, 1996), p.293; K.-U.Schoningetal, Science (2000), 290: 1347-1351; A.Eschenmoseretal, Helv.Chim.Acta (1992), 75: 218; J .Hunzikeretal, Helv.Chim.Acta (1993), 76:259; G.Ottingetal, Helv.Chim.Acta (1993), 76: 2701; K.Groebkeetal, Helv.Chim.Acta (1998), 81: 375; AndA. Eschenmoser, Science (1999), 284: 2118 is included. Modifications to the 2'modification are described in Verma, S.etal.Annu.Rev.Biochem.1998,67,99-134 and all documents described therein. Specific modifications to ribose are described in the following literature: 2'-fluoro (Kawasakiet.al., J.Med.Chem., 1993,36,831-841), 2'-MOE (Martin, P. Helv). .Chim.Acta1996,79,1930-1938), "LNA" (Wengel, J.Acc.Chem.Res.1999,32,301-310). In some embodiments, the modified sugar is any of those described in PCT Publication No. 2012/030683, the publication of which is incorporated herein by reference and of the present application. It is described in FIGS. 26 to 30.

Oligonucleotides In some embodiments, the present invention provides chirally controlled oligonucleotides and oligonucleotide compositions. For example, in some embodiments, the provided composition comprises a predetermined level of one or more individual oligonucleotide types, wherein the oligonucleotide type is 1) a base sequence; 2) a pattern of skeletal binding; 3). It is defined by the pattern of skeletal stereocenter; and 4) the pattern of skeletal P-modification.

In some embodiments, the oligonucleotide provided is a unimmer. In some embodiments, the oligonucleotide provided is a P-modified unimmer. In some embodiments, the oligonucleotide provided is a stereo-unimer. In some embodiments, the oligonucleotide provided is a stereo-unimer with an Rp configuration. In some embodiments, the oligonucleotide provided is a stereo unimmer in the Sp configuration.

In some embodiments, the oligonucleotide provided is an altomer. In some embodiments, the oligonucleotide provided is a P-modified altomer. In some embodiments, the oligonucleotide provided is a stereo altomer.

In some embodiments, the oligonucleotide provided is blockmer. In some embodiments, the oligonucleotide provided is a P-modified blocker. In some embodiments, the oligonucleotide provided is a stereoblockmer.

In some embodiments, the oligonucleotide provided is a gapmer.

In some embodiments, the oligonucleotide provided is Skipmer.

In some embodiments, the oligonucleotide provided is a combination of one or more of unimmer, altomer, blockmer, gapmer, and skipmer. For example, in some embodiments, the oligonucleotides provided are both altomers and gapmers. In some embodiments, the nucleotides provided are both gapmers and skipmers. A number of different combinations of patterns are possible, in which the components required to synthesize the oligonucleotides provided under the method according to the invention are commercially available and / or utilized for synthesis. It will be recognized by those skilled in the art of chemistry and synthesis that it is limited only by possibility.

In some embodiments, the oligonucleotides provided include one or more optionally substituted nucleotides. In some embodiments, the oligonucleotides provided include one or more modified nucleotides. In some embodiments, the oligonucleotides provided include one or more optionally substituted nucleosides. In some embodiments, the oligonucleotides provided contain one or more modified nucleosides. In some embodiments, the oligonucleotides provided include one or more optionally substituted LNAs.

In some embodiments, the oligonucleotides provided include one or more optionally substituted nucleobases. In some embodiments, the oligonucleotides provided include one or more optionally substituted native nucleobases. In some embodiments, the oligonucleotides provided include one or more optionally substituted modified nucleobases. In some embodiments, the oligonucleotides provided include one or more 5-methylcytidines; 5-hydroxymethylcytidine, 5-formylcytosine, or 5-carboxylcytosine. In some embodiments, the oligonucleotide provided comprises one or more 5-methylcytidine.

In some embodiments, the oligonucleotides provided include one or more optionally substituted sugars. In some embodiments, the oligonucleotides provided include one or more optionally substituted sugars found in native DNA and RNA. In some embodiments, the oligonucleotides provided include one or more optionally substituted ribose or deoxyribose. In some embodiments, the provided oligonucleotide comprises one or more optionally substituted ribose or deoxyribose, and one or more hydroxyl groups of the ribose or deoxyribose moiety are independently halogen, R',. It may be replaced by -N (R') ₂ , -OR' or -SR', and each R'is independently defined as described above and is as described herein. In some embodiments, the oligonucleotide provided comprises one or more optionally substituted deoxyribose, where the 2'position of deoxyribose is independently halogen, R', -N (R') ₂ . , -OR', or -SR', and each R'is independently defined as described above and is as described herein. In some embodiments, the oligonucleotide provided comprises one or more optionally substituted deoxyribose, where the 2'position of deoxyribose may be independently substituted with halogen. In some embodiments, the oligonucleotides provided contain one or more optionally substituted deoxyribose, where the 2'position of deoxyribose is independently one or more -F. It may be substituted with a halogen. In some embodiments, the oligonucleotide provided comprises one or more optionally substituted deoxyribose, where the 2'position of deoxyribose may be independently substituted with -OR', respectively. R'is independently defined as described above and is as described herein. In some embodiments, the oligonucleotide provided comprises one or more optionally substituted deoxyribose, where the 2'position of deoxyribose may be independently substituted with -OR', respectively. R'is a C ₁ to C ₆ aliphatic that may be independently substituted. In some embodiments, the oligonucleotide provided comprises one or more optionally substituted deoxyribose, where the 2'position of deoxyribose may be independently substituted with -OR', respectively. R'is a C ₁ to C ₆ alkyl that may be independently substituted. In some embodiments, the oligonucleotide provided comprises one or more optionally substituted deoxyribose, where the 2'position of deoxyribose may be independently substituted with -OMe. In some embodiments, the oligonucleotide provided comprises one or more optionally substituted deoxyribose, where the 2'position of deoxyribose may be independently substituted with -O-methoxyethyl. ..

In some embodiments, the oligonucleotide provided is a single-stranded oligonucleotide.

In some embodiments, the oligonucleotide provided is a hybridized oligonucleotide chain. In certain embodiments, the oligonucleotide provided is a partially hybridized oligonucleotide chain. In certain embodiments, the oligonucleotide provided is a fully hybridized oligonucleotide chain. In certain embodiments, the oligonucleotide provided is a double-stranded oligonucleotide. In certain embodiments, the oligonucleotide provided is a triple-stranded oligonucleotide (eg, triple-stranded).

In some embodiments, the oligonucleotides provided are chimeric. For example, in some embodiments, the oligonucleotides provided are DNA-RNA chimera, DNA-LNA chimera and the like.

In some embodiments, any one of the oligonucleotide-containing structures described in WO 2012/030683 provides a modified and chirally controlled variant thereof according to the method of the invention. For example, in some embodiments, the chiral-controlled variant comprises a stereochemical modification to any of one or more bound phosphates and / or to any of one or more bound phosphates. Includes P-modification. For example, in some embodiments, a particular nucleotide unit of an oligonucleotide of WO 2012/030683 is preselected, sterically chemically modified with the conjugated phosphate of that nucleotide unit, and / or. It is to be P-modified with the conjugated phosphate of the nucleotide unit. In some embodiments, the chirally controlled oligonucleotide is any one of the structures shown in FIGS. 26-30. In some embodiments, the chirally controlled oligonucleotide is a variant (eg, modified version) of any one of the structures shown in FIGS. 26-30. The disclosure of International Publication No. 2012/030683 is incorporated herein by reference in its entirety.

In some embodiments, the oligonucleotides provided are therapeutic agents.

In some embodiments, the oligonucleotide provided is an antisense oligonucleotide.

In some embodiments, the oligonucleotide provided is an antigene oligonucleotide.

In some embodiments, the oligonucleotide provided is a decoy oligonucleotide.

In some embodiments, the oligonucleotides provided are part of a DNA vaccine.

In some embodiments, the oligonucleotides provided are immunomodulatory oligonucleotides, such as immunostimulatory oligonucleotides and immunosuppressive oligonucleotides.

In some embodiments, the oligonucleotide provided is an adjuvant.

In some embodiments, the oligonucleotide provided is an aptamer.

In some embodiments, the oligonucleotide provided is a ribozyme.

In some embodiments, the oligonucleotide provided is a deoxyribozyme (DNAzyme or DNA enzyme).

In some embodiments, the oligonucleotide provided is small interfering RNA.

In some embodiments, the oligonucleotide provided is microRNA, or microRNA (miRNA).

In some embodiments, the provided oligonucleotides are ncRNAs (non-coding RNAs), including small non-coding RNAs such as long non-coding RNAs (lncRNAs) and Piwi-interacting RNAs (piRNAs).

In some embodiments, the oligonucleotides provided are complementary to a structural RNA, eg, tRNA.

In some embodiments, the oligonucleotides provided are nucleic acid analogs, such as GNA, LNA, PNA, TNA and morpholino.

In some embodiments, the oligonucleotide provided is a P-modified prodrug.

In some embodiments, the oligonucleotide provided is a primer. In some embodiments, primers are used for polymerase-based chain reactions (ie, PCR) to amplify nucleic acids. In some embodiments, the primers are used for any of the known variants of PCR, such as reverse transcription PCR (RT-PCR) and real-time PCR.

In some embodiments, the oligonucleotides provided are characterized by the ability to regulate RNA degrading enzyme H activation. For example, in some embodiments, RNA degrading enzyme H activation is regulated by the presence of sterically regulated phosphorothioate nucleic acid analogs, and native DNA / RNA is equivalent or more sensitive compared to the Rp stereoisomer. Rp stereoisomers are more sensitive than the corresponding Sp stereoisomers.

In some embodiments, the oligonucleotides provided are characterized by the ability to indirectly or directly increase or decrease the activity of the protein, or to suppress or promote the expression of the protein. In some embodiments, the oligonucleotides provided are characterized by their usefulness in controlling cell proliferation, viral replication, and / or other cell signaling processes.

In some embodiments, the oligonucleotides provided are about 2 to about 200 nucleotides in length. In some embodiments, the oligonucleotides provided are about 2 to about 180 nucleotides in length. In some embodiments, the oligonucleotides provided are about 2 to about 160 nucleotides in length. In some embodiments, the oligonucleotides provided are about 2 to about 140 nucleotide units in length. In some embodiments, the oligonucleotides provided are about 2 to about 120 nucleotide units in length. In some embodiments, the oligonucleotides provided are about 2 to about 100 nucleotides in length. In some embodiments, the oligonucleotides provided are about 2 to about 90 nucleotides in length. In some embodiments, the oligonucleotides provided are about 2 to about 80 nucleotides in length. In some embodiments, the oligonucleotides provided are about 2 to about 70 nucleotides in length. In some embodiments, the oligonucleotides provided are about 2 to about 60 nucleotides in length. In some embodiments, the oligonucleotides provided are about 2 to about 50 nucleotides in length. In some embodiments, the oligonucleotides provided are about 2 to about 40 nucleotides in length. In some embodiments, the oligonucleotides provided are about 2 to about 30 nucleotides in length. In some embodiments, the oligonucleotides provided are about 2 to about 29 nucleotide units in length. In some embodiments, the oligonucleotides provided are about 2 to about 28 nucleotides in length. In some embodiments, the oligonucleotides provided are about 2 to about 27 nucleotides in length. In some embodiments, the oligonucleotides provided are about 2 to about 26 nucleotides in length. In some embodiments, the oligonucleotides provided are about 2 to about 25 nucleotides in length. In some embodiments, the oligonucleotides provided are about 2 to about 24 nucleotide units in length. In some embodiments, the oligonucleotides provided are about 2 to about 23 nucleotides in length. In some embodiments, the oligonucleotides provided are about 2 to about 22 nucleotide units in length. In some embodiments, the oligonucleotides provided are about 2 to about 21 nucleotide units in length. In some embodiments, the oligonucleotides provided are about 2 to about 20 nucleotides in length.

In some embodiments, the oligonucleotides provided are about 4 to about 200 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 180 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 160 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 140 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 120 nucleotide units in length. In some embodiments, the oligonucleotides provided are about 4 to about 100 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 90 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 80 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 70 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 60 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 50 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 40 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 30 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 29 nucleotide units in length. In some embodiments, the oligonucleotides provided are about 4 to about 28 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 27 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 26 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 25 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 24 nucleotide units in length. In some embodiments, the oligonucleotides provided are about 4 to about 23 nucleotides in length. In some embodiments, the oligonucleotides provided are about 4 to about 22 nucleotide units in length. In some embodiments, the oligonucleotides provided are about 4 to about 21 nucleotide units in length. In some embodiments, the oligonucleotides provided are about 4 to about 20 nucleotides in length.

In some embodiments, the oligonucleotides provided are about 5 to about 10 nucleotides in length. In some embodiments, the oligonucleotides provided are about 10 to about 30 nucleotides in length. In some embodiments, the oligonucleotides provided are about 15 to about 25 nucleotide units in length. In some embodiments, the oligonucleotides provided are about 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22. , 23, 24, or 25 nucleotide units in length.

In some embodiments, the oligonucleotide is at least 2 nucleotide units in length. In some embodiments, oligonucleotides are at least 3 nucleotide units in length. In some embodiments, the oligonucleotide is at least 4 nucleotide units in length. In some embodiments, the oligonucleotide is at least 5 nucleotide units in length. In some embodiments, the oligonucleotide is at least 6 nucleotide units in length. In some embodiments, the oligonucleotide is at least 7 nucleotide units in length. In some embodiments, the oligonucleotide is at least 8 nucleotide units in length. In some embodiments, oligonucleotides are at least 9 nucleotide units in length. In some embodiments, oligonucleotides are at least 10 nucleotide units in length. In some embodiments, the oligonucleotide is at least 11 nucleotide units in length. In some embodiments, the oligonucleotide is at least 12 nucleotide units in length. In some embodiments, oligonucleotides are at least 15 nucleotide units in length. In some embodiments, oligonucleotides are at least 20 nucleotide units in length. In some embodiments, oligonucleotides are at least 25 nucleotide units in length. In some embodiments, oligonucleotides are at least 30 nucleotide units in length. In some embodiments, the oligonucleotide is a double strand of complementary strands with a length of at least 18 nucleotides. In some embodiments, the oligonucleotide is a double strand of complementary strands with a length of at least 21 nucleotides.

In some embodiments, the 5'end and / or 3'end of the provided oligonucleotide is modified. The 5'end and / or 3'end of the oligonucleotides provided in some embodiments are modified with a terminal cap moiety. Such exemplary modifications, including end cap moieties, are described in detail herein and in the art, eg, but not limited to, US Patent Application Publication No. 2009/0023675A1.

Species of Oligonucleotides In certain embodiments, the oligonucleotide of formula I is one of the structures shown in Table 2 above or described in Examples.

In some embodiments, the chiral controlled oligonucleotides provided comprise a sequence of mipomersen, or a portion of the sequence of mipomersen. Mipomersen is based on the following nucleotide sequence GCCT / UCAGT / UCT / UGCT / UT / UCGCACC. In some embodiments, either one or more nucleotides or bonds may be modified according to the invention. In some embodiments, the present invention has the following sequence: G * -C * -C * -U * -C * -dA-dG-dT-dC-dT-dG with a 3'→ 5'phospholothioate binding. Chiral controlled with -dmC-dT-dT-dmC-G * -C * -A * -C * -C * [d = 2'-deoxy, * = 2'-O- (2-methoxyethyl)] Also provides an oligonucleotide. Exemplary modified mipomersen sequences are described throughout this application and include, but are not limited to, those listed in Table 4.

In certain embodiments, the oligonucleotide provided is Juni Mar. In certain embodiments, the oligonucleotide provided is a Mipomersen unimmer with an Rp configuration. In certain embodiments, the oligonucleotide provided is a Sp-arranged Mipomersen unimmer.

Exemplary chirally controlled oligonucleotides containing the sequence of mipomersen, or a portion of the sequence of mipomersen, are listed in Table 4 below.

Exemplary mipomersen-related sequences

Oligonucleotide Compositions The present invention provides a composition comprising a plurality of provided oligonucleotides or a composition comprising a plurality of provided oligonucleotides (eg, a chiral-controlled oligonucleotide composition). In some embodiments, such provided oligonucleotides are all of the same type, i.e., all of the same base sequence, pattern of skeletal binding (ie, pattern of internucleotide binding type, eg, phosphate, etc.). Phosphate (such as phosphorothioate), skeletal chiral center patterns (ie, patterns of bound phosphate stereochemistry (Rp / Sp)), and patterns of phosphate skeletal modification (eg, "-XLR ¹ " groups of the pattern of formula I). Has. However, in many embodiments, the provided composition generally comprises a predetermined relative amount of a plurality of oligonucleotide types.

In some embodiments, the chiral controlled oligonucleotide composition provided is a chirally pure mipomersen composition. That is, in some embodiments, the chiral controlled oligonucleotide compositions provided provide mipomersen as one diastereomer with respect to the arrangement of bound phosphates.

In some embodiments, the chirally controlled oligonucleotide composition provided is a chirally uniform mipomersen composition. That is, in some embodiments, each and every bound phosphate of mipomersen is present in the Rp configuration, or each and every bound phosphate of mipomersen is present in the Sp configuration.

In some embodiments, the provided chiral controlled oligonucleotide compositions comprise one or more combinations of the provided oligonucleotide types. It will be appreciated by those skilled in the art of chemistry and medicine that the selection and amount of each type of one or more of the oligonucleotides provided in the provided composition will depend on the intended use of the composition. That is, one of ordinary skill in the art will appreciate that the amount and type contained in the oligonucleotide provided will have certain desirable properties (eg, biologically favorable properties, therapeutically favorable properties, etc.) for the composition as a whole. Will be designed to provide chiral controlled oligonucleotide compositions.

In some embodiments, the chiral controlled oligonucleotide compositions provided are a combination of two or more provided oligonucleotide types. In some embodiments, the chiral controlled oligonucleotide compositions provided are a combination of three or more provided oligonucleotide types. In some embodiments, the chiral controlled oligonucleotide compositions provided are a combination of four or more provided oligonucleotide types. In some embodiments, the chiral controlled oligonucleotide compositions provided are a combination of 5 or more provided oligonucleotide types. In some embodiments, the chiral controlled oligonucleotide compositions provided are a combination of 6 or more provided oligonucleotide types. In some embodiments, the chiral controlled oligonucleotide compositions provided are a combination of seven or more provided oligonucleotide types. In some embodiments, the chiral controlled oligonucleotide compositions provided are a combination of eight or more provided oligonucleotide types. In some embodiments, the chiral controlled oligonucleotide compositions provided are a combination of nine or more provided oligonucleotide types. In some embodiments, the chiral controlled oligonucleotide compositions provided are a combination of 10 or more provided oligonucleotide types. In some embodiments, the chiral controlled oligonucleotide compositions provided are a combination of 15 or more provided oligonucleotide types.

In some embodiments, the chiral-controlled oligonucleotide compositions provided are a combination of a quantification of chirally homogeneous mipomersen with an Rp configuration and a quantification of chirally uniform mipomersen with a Sp configuration.

In some embodiments, the chiral controlled oligonucleotide compositions provided are a quantification of chirally homogeneous mipomersen with an Rp configuration, a quantification of chirally uniform mipomersen with a Sp configuration, and the desired diastereomer. A quantitative combination of one or more chirally pure mipomersen in stereomeric form.

Methods for Making Chirally Controlled Oligonucleotides and Compositions The Invention The present invention provides methods for making chirally controlled oligonucleotides and chirally controlled compositions that include one or more specific nucleotide types. As mentioned above, the term "oligonucleotide type" herein refers to a particular base sequence, skeletal binding pattern, skeletal chiral center pattern, and phosphate skeletal modification pattern (eg, "-XLR ¹ " group). Define the oligonucleotide to have. Commonly specified "type" oligonucleotides are structurally identical to each other with respect to base sequences, skeletal binding patterns, skeletal chiral center patterns, and phosphate skeletal modification patterns.

In some embodiments, the chirally controlled oligonucleotides provided in the present invention have different properties than those corresponding to sterically random oligonucleotide mixtures. In some embodiments, the chirally controlled oligonucleotide has a lipophilicity different from that of a sterically random oligonucleotide mixture. In some embodiments, chirally controlled oligonucleotides have different retention times on HPLC. In some embodiments, the chirally controlled oligonucleotide may have a peak retention time that is significantly different from that corresponding to a sterically random oligonucleotide mixture. During oligonucleotide purification using HPLC, as is commonly done in the art, certain chirally controlled oligonucleotides are largely, if not completely, lost. Certain chirally controlled oligonucleotides are lost, if not completely, during oligonucleotide purification using HPLC, as is commonly done in the art. One result is that certain diastereomers, a sterically random oligonucleotide mixture, are not tested in the assay. Another result is that, for each batch, sterically random oligonucleotides that are presumed to be "pure" due to unavoidable instrumental and human error are the diastereomers in the composition and their relative amounts as well. It will contain inconsistent compositions in that the absolute amount varies from batch to batch. Because the chiral-controlled oligonucleotide is synthesized as a diastereomer in a chiral-controlled manner, the chiral-controlled oligonucleotide composition comprises a predetermined level of one or more individual oligonucleotide types. The chiral-controlled oligonucleotides and chiral-controlled oligonucleotide compositions provided in the present invention overcome such problems.

Those skilled in the art of chemistry and synthesis provide some control during each step of the synthesis of the provided oligonucleotides in the synthetic methods of the invention, with phosphoric acid to which each nucleotide unit of the oligonucleotide is pre-bound. Recognize that specific chemistries and / or specific modifications with bound phosphate and / or can be designed and / or selectable to have specific bases and / or specific sugars. Will. In some embodiments, the oligonucleotides provided are pre-designed and / or selected to have a particular combination of stereocenters in the phosphate bound by the internucleotide linkage.

In some embodiments, the oligonucleotides made and provided using the methods of the invention are designed and / or determined to have a particular combination of bound phosphate modifications. In some embodiments, the oligonucleotides made and provided using the methods of the invention are designed and / or determined to have a particular combination of bases. In some embodiments, the oligonucleotides made and provided using the methods of the invention are designed and / or determined to have a particular combination of sugars. In some embodiments, the oligonucleotides made and provided using the methods of the invention are designed and / or determined to have a particular combination of one or more of the above structural properties.

The method of the present invention exhibits a high degree of chiral control. For example, the methods of the invention facilitate control of the stereochemical arrangement of each single bound phosphate within the provided oligonucleotide. In some embodiments, the methods of the invention provide oligonucleotides containing one or more modified internucleotide linkages that independently have the structure of Formula I.

In some embodiments, the methods of the invention provide oligonucleotides that are mipomersen unimmers. In some embodiments, the methods of the invention provide oligonucleotides that are Mipomersen unimmers in the Rp configuration. In some embodiments, the methods of the invention provide oligonucleotides that are Sp-arranged Mipomersen unimmers.

In some embodiments, the methods of the invention provide chirally controlled oligonucleotide compositions, i.e., oligonucleotide compositions comprising a given level of individual oligonucleotide types. In some embodiments, the chirally controlled oligonucleotide composition comprises one oligonucleotide type. In some embodiments, the chirally controlled oligonucleotide composition comprises more than one oligonucleotide type. In some embodiments, the chirally controlled oligonucleotide composition comprises a plurality of oligonucleotide types. Exemplary chirally controlled oligonucleotide compositions made according to the present invention are as described herein.

In some embodiments, the methods of the invention provide a chirally pure mipomersen composition with respect to the arrangement of bound phosphoric acid. Thus, in some embodiments, the methods of the invention provide a composition of mipomersen in which mipomersen is present in the composition in the form of a single diastereomer with respect to the arrangement of bound phosphate.

In some embodiments, the methods of the invention provide a chirally uniform mipomersen composition with respect to the arrangement of bound phosphoric acid. Thus, in some embodiments, the method of the invention provides a composition of mipomersen having the same steric chemistry with respect to the arrangement of phosphate to which all nucleotide units are bound, eg, all nucleotide units are bound. A composition in which the Rp configuration is in the phosphoric acid, or where all nucleotide units are in the Sp configuration in the bound phosphoric acid.

In some embodiments, the chiral controlled oligonucleotides provided are 50% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 55% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 60% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 65% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 70% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 75% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 80% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 85% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 90% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 91% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 92% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 93% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 94% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 95% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 96% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 97% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 98% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 99% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 99.5% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 99.6% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 99.7% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 99.8% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are approximately 99.9% ultrapure. In some embodiments, the chiral controlled oligonucleotides provided are at least about 99% pure.

In some embodiments, the chirally controlled oligonucleotide composition is a composition designed to contain one oligonucleotide type. In certain embodiments, such compositions are approximately 50% diastereomerically pure. In some embodiments, such compositions are approximately 50% diastereomerically pure. In some embodiments, such compositions are approximately 50% diastereomerically pure. In some embodiments, such compositions are approximately 55% diastereomerically pure. In some embodiments, such compositions are approximately 60% diastereomerically pure. In some embodiments, such compositions are approximately 65% diastereomerically pure. In some embodiments, such compositions are approximately 70% diastereomerically pure. In some embodiments, such compositions are approximately 75% diastereomerically pure. In some embodiments, such compositions are approximately 80% diastereomerically pure. In some embodiments, such compositions are approximately 85% diastereomerically pure. In some embodiments, such compositions are approximately 90% diastereomerically pure. In some embodiments, such compositions are approximately 91% diastereomerically pure. In some embodiments, such compositions are approximately 92% diastereomerically pure. In some embodiments, such compositions are approximately 93% diastereomerically pure. In some embodiments, such compositions are approximately 94% diastereomerically pure. In some embodiments, such compositions are approximately 95% diastereomerically pure. In some embodiments, such compositions are approximately 96% diastereomerically pure. In some embodiments, such compositions are approximately 97% diastereomerically pure. In some embodiments, such compositions are approximately 98% diastereomerically pure. In some embodiments, such compositions are approximately 99% diastereomerically pure. In some embodiments, such compositions are approximately 99.5% diastereomerically pure. In some embodiments, such compositions are approximately 99.6% diastereomerically pure. In some embodiments, such compositions are approximately 99.7% diastereomerically pure. In some embodiments, such compositions are approximately 99.8% diastereomerically pure. In some embodiments, such compositions are approximately 99.9% diastereomerically pure. In some embodiments, such compositions are at least about 99% diastereomerically pure.

In some embodiments, the chirally controlled oligonucleotide composition is a composition designed to include multiple oligonucleotide types. In some embodiments, the methods of the invention allow the generation of a library of chirally controlled oligonucleotides, with any one or more preselected amounts of any one or more chiral controlled oligonucleotide types. Can be mixed with another chirally controlled oligonucleotide type of to make a chirally controlled oligonucleotide composition. In some embodiments, a preselected amount of oligonucleotide type is a composition having any one of the above-mentioned diastereomeric purity.

In some embodiments, the invention is described in the following steps:
(1) Coupling;
(2) Capping;
(3) Modification;
Provided is a chirally controlled oligonucleotide production method comprising repeating steps (1) to (4) until (4) deblocking; and (5) the desired length is achieved.

When describing the methods provided, the term "cycle" has the usual meaning understood by one of ordinary skill in the art. In some embodiments, the cycle of steps (1) to (4) is called a cycle.

In some embodiments, the invention is described in the following steps:
A chiral-controlled oligonucleotide comprising (a) providing a quantification of a first chiral-controlled oligonucleotide; and (b) optionally providing a quantification of one or more further chiral-controlled oligonucleotides. A method for producing a composition is provided.

In some embodiments, the first chirally controlled oligonucleotide is the oligonucleotide type described herein. In some embodiments, one or more further chirally controlled oligonucleotides are one or more oligonucleotide types described herein.

Those skilled in the art of related chemistry and synthesis, when synthesized using the methods of the invention, the oligonucleotides provided have certain versatility, with respect to structural deformations and stereochemical arrangements. You will understand that control is possible. For example, after the completion of the first cycle, it is possible to perform the next cycle using individually selected nucleotide units for the next cycle, and in some embodiments, the first cycle. Contains nucleobases and / or sugars that are different from the nucleobases and / or sugars of. Similarly, the chiral auxiliary used in the coupling step of the subsequent cycle may be different from the chiral auxiliary used in the first cycle, in which the second cycle has a different stereochemical arrangement. Generates a phosphate bond. In some embodiments, the stereochemistry of the newly formed phosphate bond is controlled using stereochemically pure phosphoramidite. In addition, the modifier used in the modification step of the subsequent cycle may differ from the modifier used in the first or previous cycle. The cumulative effect of this iterative construction approach is that each component of the oligonucleotide provided can be structurally and constitutively highly tailored. A further advantage of this approach is the capping step that minimizes the formation of "n-1" impurities. In the absence of capping steps, isolation of the provided oligonucleotides would be very difficult, especially for long oligonucleotides.

は、固体支持体を示し、固体支持体と結合した成長するキラル制御されたオリゴヌクレオチドの一部分であっても良い。例示的なキラル補助剤は、式３－Ｉ：

の構造を有し、
さらなる詳細は以下に記載される。「キャップ」は、キャッピングステップで窒素原子に導入される化学部分であり、いくつかの実施形態において、アミノ保護基である。第１サイクルでは、開始時に固体支持体に結合したヌクレオシドは、１つのみである可能性があるが、脱ブロッキングの前にサイクルの終了を行ってもよいことを、当業者は理解する。当業者により理解される通り、Ｂ^ＰＲＯは、オリゴヌクレオチド合成で使用される保護基である。スキームIの上記のサイクルの各ステップは、さらに以下に記載される。 In some embodiments, an exemplary cycle of methods for making chirally controlled oligonucleotides is described in Scheme I. In Scheme I

Represents a solid support and may be part of a growing chiral controlled oligonucleotide bound to the solid support. An exemplary chiral auxiliary is of formula 3-I:

Has the structure of
Further details are given below. A "cap" is a chemical moiety introduced into a nitrogen atom during the capping step and, in some embodiments, is an amino protecting group. Those skilled in the art will appreciate that in the first cycle, there may be only one nucleoside bound to the solid support at the beginning, but the end of the cycle may occur prior to deblocking. As will be appreciated by those skilled in the art, B ^PRO is a protecting group used in oligonucleotide synthesis. Each step of the above cycle of Scheme I is further described below.

Scheme I. Synthesis of chirally controlled oligonucleotides

Synthesis on Solid Supports In some embodiments, the synthesis of the provided oligonucleotides takes place on a solid phase. In some embodiments, the reactive groups present on the solid support are protected. In some embodiments, the reactive groups present on the solid support are unprotected. During oligonucleotide synthesis, the solid support is treated with various reagents in several synthesis cycles to achieve gradual elongation of oligonucleotide chains that grow on an individual nucleotide basis. The nucleoside unit at the end of the chain that binds directly to the solid support is referred to herein as the "first nucleoside". The first nucleoside is attached to the solid support by a covalent bond between the CPG, polymer or another solid support and the nucleoside through a linker moiety, i.e. a diradical. The linker remains intact during the synthetic cycle of building the oligonucleotide chain and is cleaved after chain building to release the oligonucleotide from the support.

Solid supports for solid phase nucleic acid synthesis include, for example, U.S. Pat. Nos. 4,659,774, 5,141,813, 4,458,066; U.S. Pat. No. 4,415,732 of Caruthers. , 4,458,066, 4,500,707, 4,668,777, 4,973,679, and 5,132,418; U.S. Pat. No. 5, 132,418 of Andrus et al. Includes the supports described in 047,524, 5,262,530; and Koster's US Pat. No. 4,725,677 (reissued as Re34,069). In some embodiments, the solid phase is an organic polymer support. In some embodiments, the solid phase is an inorganic polymer support. In some embodiments, the organic polymer support is polystyrene, aminomethylpolystyrene, polyethylene glycol-polystyrene graft copolymer, polyacrylamide, polymethacrylate, polyvinyl alcohol, highly crosslinked polymer (HCP), or another. Includes synthetic polymers of, cellulose and starches or carbohydrates such as another polymeric carbohydrate, or other organic polymers and copolymers, composite materials or combinations of the above inorganic or organic materials. In some embodiments, the inorganic polymer support is a controlled polyglass (CPG) such as silica, alumina, silica gel support, or aminopropyl CPG. Other useful solid supports include fluorolas solid supports (see, eg WO / 2005/070859), long chain alkylamine (LCAA) controlled porous glass (CPG) solid supports (eg, SP Adams, KS Kavka). , EJ Wykes, SB Holder and GR Galluppi, J. Am. Chem. Soc., 1983, 105, 661-663; GR Gough, MJ Bruden and PT Gilham, Tetrahedron Lett., 1981, 22, 4177-4180) Can be mentioned. Membrane supports and polymer membranes (see, eg, Innovation and Perspectives in Solid Phase Synthesis, Peptides, Proteins and Nucleic Acids, ch 21 pp 157-162, 1994, Ed. Roger Epton and US Pat. No. 4,923,901). It is also useful for nucleic acid synthesis. Once formed, the membrane can be chemically functionalized for use in nucleic acid synthesis. In addition to the binding of functional groups to the membrane, the use of linker or spacer groups bound to the membrane is used to minimize steric hindrance between the membrane and the synthetic chain.

Other preferred solid supports include those commonly known in the art and used in solid phase methods, such as glass sold as PolymerTM 200 support, controlled polystyrene (CPG), oxalyl-. Controlled porous glass (see, eg, Alul, et al., Nucleic Acids Research, 1991, 19, 1527), TentaGel support-aminopolyethylene glycol inducible support (eg, Wright, et al., Tetrahedron Lett., 1993, 34, 3373) and Poros-polystyrene / divinylbenzene copolymers.

Surface-activated polymers have been utilized in the synthesis of natural and modified nucleic acids and proteins on several solid support media. The material of the solid support may be any polymer, how it accompanies with uniform porosity, sufficient amine content, and sufficient flexibility without loss of integrity. Those that can withstand various operations are preferable. Examples of materials of choice are preferably nylon, polypropylene, polyester, polytetrafluoroethylene, polystyrene, polycarbonate, and nitrocellulose. Another material may function as a solid support, depending on the researcher's design. Considering some designs, for example, a metal coated with gold or platinum in particular may be selected (see, eg, US Publication No. 20010055761). In one embodiment of oligonucleotide synthesis, for example, the nucleoside is immobilized on a solid support functionalized with hydroxyl or amino residues. The solid support also provides a derivatized, acid-labile trialkoxytrityl group, such as a trimethoxytrityl group (TMT). Without being bound by theory, the presence of a trialkoxytrityl protecting group is expected to allow early detrityling under the conditions commonly used in DNA synthesizers. For faster separation of oligonucleotide materials in aqueous ammonia, a diglycoate linker may be introduced onto the support.

In some embodiments, the oligonucleotides provided are, as an alternative, synthesized in the 5'-3'direction. In some embodiments, the nucleic acid is attached to a solid support through the 5'end of the growing nucleic acid, thereby presenting 3'groups for the reaction. That is, the reaction occurs with or during the enzymatic reaction with 5'-nucleoside phosphoramidite (eg, ligation and polymerization with nucleoside 5'-triphosphate). When considering 5'-3'synthesis, the iterative steps of the invention do not change (ie, capping and modifying chiral phosphate).

Bonding moiety A binding moiety or linker may be used to attach the solid support to a compound containing a free nucleophilic moiety. Suitable linkers, such as short molecules that function to attach a solid support to a functional group (eg, a hydroxyl group) of an early nucleoside molecule in solid phase synthesis techniques are known. In some embodiments, the binding moiety is a succinamide acid linker, or succinic acid linker (-CO-CH ₂ -CH ₂ -CO-), or an oxalyl linker (-CO-CO-). In some embodiments, the binding moiety and the nucleoside are bound together via an ester bond. In some embodiments, the binding moiety and the nucleoside bind together via an amide bond. In some embodiments, the binding moiety binds the nucleoside to another nucleotide or nucleic acid. Preferred linkers disclosed are, for example, Oligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed., IRL Press, NY, 1991, Chapter 1 and Solid-Phase Supports for Oligonucleotide Synthesis, Pon, RT, Curr. Prot. Nucleic. Described in Acid Chem., 2000, 3.1.1-3.1.28.

The linker moiety is used to attach a compound containing a free nucleophilic moiety to another nucleoside, nucleotide, or nucleic acid. In some embodiments, the binding moiety is a phosphodiester bond. In some embodiments, the binding moiety is an H-phosphonate moiety. In some embodiments, the binding moiety is a modified phosphate bond described herein. In some embodiments, a universal linker (UnyLinker) is used to attach an oligonucleotide to a solid support (Ravikumar et al., Org. Process Res. Dev., 2008, 12 (3). ), 399-410). In some embodiments, another universal linker is used (Pon, R.T., Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28). In some embodiments, various orthogonal linkers (eg, disulfide linkers) are used (Pon, R.T., Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28).

General Conditions-Solvents Used for Synthesis Synthesis of provided oligonucleotides is generally carried out in an aprotic organic solvent. In some embodiments, the solvent is carried out in a nitrile solvent such as acetonitrile. In some embodiments, the solvent is a basic amine solvent such as, for example, pyridine. In some embodiments, the solvent is an ether solvent such as, for example, tetrahydrofuran. In some embodiments, the solvent is a halogenated hydrocarbon such as, for example, dichloromethane. In some embodiments, a mixture of solvents is used. In certain embodiments, the solvent is a mixture of one or more solvents of the above classification.

In some embodiments, if the aprotic organic solvent is not basic, the base is present in the reaction step. In some embodiments, if a base is present, the base is an amine base, for example, pyridine, quinoline, or N, N-dimethylaniline. Exemplary alternative amine bases include pyrrolidine, piperidine, N-methylpyrrolidin, pyridine, quinoline, N, N-dimethylaminopyridine (DMAP), or N, N-dimethylaniline.

In some embodiments, the base is other than an amine base.

In some embodiments, the aprotic organic solvent is anhydrous. In some embodiments, the anhydrous aprotic organic solvent is freshly distilled. In some embodiments, the freshly distilled anhydrous aprotic organic solvent is a basic amine solvent such as, for example, pyridine. In some embodiments, the freshly distilled anhydrous aprotic organic solvent is an ether solvent such as, for example, tetrahydrofuran. In some embodiments, the freshly distilled anhydrous aprotic organic solvent is, for example, a nitrile solvent such as acetonitrile.

Chiral Reagents In the methods provided, chiral reagents are used to confer stereoselectivity in the production of chiral-controlled oligonucleotides. As a chiral auxiliary, many different chiral reagents cited herein by one of ordinary skill in the art may be used by the method of the invention. Such exemplary chiral reagents are as described herein and above, Wada I, II and III. In certain embodiments, the chiral reagents are as described in Wada I. In some embodiments, the chiral reagent used by the method of the invention is the following formula 3-I :.

であり、
ここでＷ^１およびＷ^２は、－Ｏ－、－Ｓ－、または－ＮＧ^５－のいずれかであり、Ｕ_１およびＵ_３は、単結合、二重結合または三重結合を通じて、Ｕ_２が存在する場合、Ｕ_２と結合する炭素原子、またはｒが、０の場合、互いに結合する炭素原子である。Ｕ_２は、－Ｃ－、－ＣＧ^８－、－ＣＧ^８Ｇ^８－、－ＮＧ^８－、－Ｎ－、－Ｏ－、または－Ｓ－であり、ｒは、０～５の整数であり、２個を超えるヘテロ原子が隣接することはない。Ｕ_２のいずれか１つがＣである場合、三重結合が、Ｃである第２の事例のＵ_２の間に形成されなければならない、またはＵ_１もしくはＵ_３のいずれか１つに形成されなければならない。同様に、Ｕ_２のいずれか１つがＣＧ^８である場合、二重結合が、－ＣＧ^８－もしくは－Ｎ－である第２の事例のＵ_２の間に形成される、または、Ｕ_１もしくはＵ_３のいずれか１つに形成される。

And
Where W ¹ and W ² are either -O-, -S-, or -NG ^5- , and U ₁ and U ₃ have U ₂ present through a single bond, double bond or triple bond. If so, it is a carbon atom bonded to U ₂ , or if r is 0, it is a carbon atom bonded to each other. U ₂ is -C-, -CG ^8- , -CG ⁸ G ^8- , -NG ^8- , -N-, -O-, or -S-, and r is an integer of 0 to 5. No more than two heteroatoms are adjacent. If any one of U ₂ is C, a triple bond must be formed between U ₂ in the second case of C, or it must be formed in either U ₁ or U ₃ . Must be. Similarly, if any one of U ₂ is CG ⁸ , a double bond is formed between U ₂ in the second case of -CG ^8- or -N-, or U ₁ or It is formed in any one of _U3 .

In some embodiments, −U ₁ − (U ₂ ) _r − U ₃ − is −CG ³ G ⁴ − CG ¹ G ² −. In some embodiments, −U ₁ − (U ₂ ) _r − U ₃ − is −CG ³ = CG ¹ −. In some embodiments, −U ₁ − (U ₂ ) _r − U ₃ − is −C≡C−. In some embodiments, −U ₁ − (U ₂ ) _r − U ₃ − is −CG ³ = CG ⁸ −CG ¹ G ² −. In some embodiments, −U ₁ − (U ₂ ) _r − U ₃ − is −CG ³ G ⁴ − O—CG ¹ G ² −. In some embodiments, -U _1- (U ₂ ) _r -U ₃ -is-CG ³ G ⁴ -NG ⁸ -CG ¹ G ^2- . In some embodiments, -U _1- (U ₂ ) _r -U _3- is -CG ³ G ⁴ -N-CG ^2- . In some embodiments, −U ₁ − (U ₂ ) _r − U ₃ − is −CG ³ G ⁴ − N = CG ⁸ − CG ¹ G ² −.

As defined herein, G ¹ , G ² , G ³ , G ⁴ , G ⁵ , and G ⁸ are independently hydrogen, or alkyl, aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heteroaryl, and. It is an arbitrarily substituted group selected from aryls; or the ^two G1 ^, G2, ^G3 , ^G4 , and ^G5 and ^G6 may be substituted together. It forms a saturated, partially unsaturated or unsaturated carbocyclic ring, or a monocyclic or polycyclic ring containing a heteroatom of up to about 20 ring atoms, which is a fused or uncondensed ring. Is. In some embodiments, the rings so formed are substituted with oxo, thioxo, alkyl, alkenyl, alkynyl, heteroaryl, or aryl moieties. In some embodiments, when a ring formed by combining two ^G6s is replaced, it is replaced by a moiety having sufficient bulk to confer stereoselectivity during the reaction. ..

In some embodiments, the ring formed by the two ^G6s together is cyclopentyl, pyrrolyl, cyclopropyl, cyclohexenyl, cyclopentenyl, tetrahydropyranyl, or piperazinyl which may be substituted. .. In some embodiments, the ring formed by combining two ^G6s may be substituted cyclopentyl, pyrrolyl, cyclopropyl, cyclohexenyl, cyclopentenyl, tetrahydropyranyl, pyrrolidinyl, or piperazinyl. Is.

In some embodiments, G ¹ is a optionally substituted phenyl. In some embodiments, G ¹ is phenyl. In some embodiments, G ² is methyl or hydrogen. In some embodiments, G ¹ is a optionally substituted phenyl and G ² is a methyl. In some embodiments, G ¹ is phenyl and G ² is methyl.

In some embodiments, r is 0.

In some embodiments, W ¹ is -NG ^5- . ^In some embodiments, any one of ^G3 and G4 forms a pyrrolidinyl ring that may be substituted with ^G5 . In some embodiments, any one of G ³ and G ⁴ together with G ⁵ forms a pyrrolidinyl ring.

In some embodiments, W ² is —O—.

ここで、各変数は、独立して、上記のとおり定義され、ここに記載したとおりである。 In some embodiments, the chiral reagent is a compound of formula 3-AA:

Here, each variable is independently defined as described above and is as described herein.

In some embodiments of formula 3AA, W ¹ and W ² are independently -NG ^5- , -O-, or -S-; G ¹ , G ² , G ³ , G ⁴ , and G ⁵ are , Independently hydrogen, or an optionally substituted group selected from alkyl, aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heteroaryl, and aryl; or two G ¹ , G ² , G ³ , G ⁴ , and G ⁵ and G ⁶ may be substituted together, saturated, partially unsaturated or unsaturated carbocyclic, or monocyclic or polycyclic maximum. It forms a ring containing a heteroatom of about 20 ring atoms, which is a fused or non-fused ring, where G ¹ , G ² , G ³ , G ⁴ , and G ⁵ and G ⁶ are 4. Never exceed. Similar to Formula 3-I, any of G ¹ , G ² , G ³ , G ⁴ , or G ⁵ may be substituted with oxo, tioxo, alkyl, alkenyl, alkynyl, heteroaryl, or aryl moieties. good. In some embodiments, such substitutions result in stereoselectivity in the production of chirally controlled oligonucleotides.

のいずれか１つである。 In some embodiments, the chiral reagent has the following formula:

It is any one of.

In some embodiments, the chiral reagent is an amino alcohol. In some embodiments, the chiral reagent is aminothiol. In some embodiments, the chiral reagent is aminophenol. In some embodiments, the chiral reagents are (S)-and (R) -2-methylamino-1-phenylethanol, (1R, 2S) -ephedrine, or (1R, 2S) -2-methylamino-. 1,2-diphenylethanol.

いずれか１つの化合物である。 In some embodiments of the invention, the chiral reagent has the following formula:

Any one compound.

The choice of chiral reagent, eg, the isomer of formula Q, or its stereoisomer of formula R, is the bound phosphoric acid that allows for unique control of chirality. Thus, either the Rp or Sp configuration is selectable in each synthetic cycle, allowing control over the entire three-dimensional structure of the chirally controlled oligonucleotide. In some embodiments, chirally controlled oligonucleotides all have an Rp stereocenter. In some embodiments of the invention, the chirally controlled oligonucleotides all have a Sp stereocenter. In some embodiments of the invention, each bound phosphate of the chirally controlled oligonucleotide is independently Rp or Sp. In some embodiments of the invention, each bound phosphate of the chirally controlled oligonucleotide is independently Rp or Sp, and at least one Rp, and at least one Sp. In some embodiments, the selection of Rp and Sp centers is done so as to confer a specific three-dimensional superstructure to the chirally controlled oligonucleotide. Such exemplary selections are described in more detail herein.

In some embodiments, the chiral reagent used according to the invention is selected by its ability to be removed in a step during the cycle described above. For example, in some embodiments, it is desirable to remove the chiral reagent during the modification step of the bound phosphate. In some embodiments, it is desirable to remove the chiral reagent prior to the modification step of the bound phosphate. In some embodiments, it is desirable to remove the chiral reagent after the modification step of the bound phosphate. In some embodiments, after performing the first coupling step and after performing the first coupling step so that the chiral reagent is not present on the growing oligonucleotide during the second coupling, and in the second coupling step. It is desirable to remove the chiral reagent before performing (and so on for the subsequent coupling steps). In some embodiments, the chiral reagent is removed during a "deblocking" reaction that is performed after modification of the bound phosphate but before the onset of the subsequent cycle. Exemplary methods and reagents for removal are described herein.

In some embodiments, removal of the chiral auxiliary is achieved when performing the modification and / or deblocking steps described in Scheme I. It is beneficial to combine chiral auxiliary removal with other transformations such as modification and deblocking. Those skilled in the art will appreciate that by omitting step / conversion, the overall efficiency of synthesis can be increased, for example, especially with respect to longer oligonucleotide yields and product purity. An example in which the chiral auxiliary is removed during modification and / or deblocking is described in Scheme I.

In some embodiments, the chiral reagents used by the methods of the invention are characterized by being removable under certain conditions. For example, in some embodiments, the chiral reagent is selected by its ability to be removed under acidic conditions. In certain embodiments, the chiral reagent is selected by its ability to be removed under weakly acidic conditions. In certain embodiments, the chiral reagent is selected according to its ability to be removed by the method of the E1 removal reaction (eg, under acidic conditions, a cation intermediate is formed on the chiral reagent to remove the chiral reagent. Separate from oligonucleotides). In some embodiments, the chiral reagent is characterized by having a structure that is recognized as capable of adapting to or facilitating an E1 removal reaction. Those skilled in the art will appreciate that it is possible to foresee which structures will tend to withstand such removal reactions.

In some embodiments, the chiral reagent is selected by its ability to be removed with a nucleophile. In some embodiments, the chiral reagent is selected by its ability to be removed with an amine nucleophile. In some embodiments, the chiral reagent is selected by its ability to be removed using a nucleophile other than an amine.

In some embodiments, the chiral reagent is selected by its ability to be removed with a base. In some embodiments, the chiral reagent is selected by its ability to be removed with an amine. In some embodiments, the chiral reagent is selected by its ability to be removed with a base other than an amine.

Further Embodiments of Chiral Reagents In some embodiments, the present invention relates to chiral reagents used to synthesize chiral controlled oligonucleotides.

In some embodiments, the invention provides a stable chiral reagent for each of the coupling, capping, modification, and deblocking steps described above and herein. In some embodiments, the invention provides a chiral reagent that is stable for each of the modifications and deblocking steps described above and herein. In some embodiments, the invention provides a chiral reagent that is stable for each of the sulfurization and deblocking steps described above and herein. In some embodiments, the invention provides a chiral reagent that is stable to the steps of oxidation described above and herein. In some embodiments, such chiral reagents have the structure of formula Z-I.

In some embodiments, the invention provides a chiral reagent that is removed by treatment with a base and / or nucleophile. In some embodiments, the invention is removed by treatment with bases and / or nucleophiles and is stable to the coupling, capping, modification, and deblocking steps described above and herein. To provide a sexual chiral reagent. In some embodiments, the invention provides a chiral reagent that is removed by a treatment containing an amine. In some embodiments, the invention is a chiral reagent that is removed by treatment with amines and is stable to each of the coupling, capping, modification, and deblocking steps described above and herein. I will provide a. In some embodiments, the invention is removed by the deprotection / cutting conditions described herein and for each of the coupling, capping, modification, and deblocking steps described above and herein. A stable chiral reagent is provided. In some embodiments, such chiral reagents have the structure of formula Z-I.

In some embodiments, chiral reagents that are stable for each step of coupling, capping, modification, and deblocking are used to synthesize the chiral controlled oligonucleotides described above and herein. In some embodiments, chiral reagents that are stable for each step of coupling, capping, modification, and deblocking are used to synthesize the chiral controlled oligonucleotides described above and herein. Chiral control nucleotides include one or more phosphate diester bonds or phosphodiester bonds. In some embodiments, a chiral control comprising one or more phosphate diester bonds or phosphodiester bonds using a chiral reagent that is stable for each step of coupling, capping, modification, and deblocking. The oligonucleotide is synthesized and the reagent is not removed until the oligonucleotide reaches the desired length. In some embodiments, a chiral control comprising one or more phosphate diester bonds or phosphodiester bonds using a chiral reagent that is stable for each step of coupling, capping, modification, and deblocking. Oligonucleotides are synthesized and reagents are not removed until after the end of the cycle. In some embodiments, a chiral control comprising one or more phosphate diester bonds or phosphodiester bonds using a chiral reagent that is stable for each step of coupling, capping, modification, and deblocking. Oligonucleotides are synthesized and the reagents are not removed until cleavage from the solid support. In some embodiments, a chiral control comprising one or more phosphate diester bonds or phosphodiester bonds using a chiral reagent that is stable for each step of coupling, capping, modification, and deblocking. The oligonucleotide is synthesized and the reagent is not removed until cleavage from the solid support, removal is performed in the same steps as cleavage from the solid support. In some embodiments, such chiral reagents have the structure of formula Z-I.

In some embodiments, when a chiral reagent stable for each step of coupling, capping, modification, and deblocking is used in oligonucleotide synthesis, it has 5'-OH ready for coupling. Oligonucleotides may be from any synthetic cycle, including those described in Schemes I, Ib, Ic, Id, Z-1 and Z-2. In some embodiments, oligonucleotides with 5'-OH that are ready for coupling include various types of internucleotide linkages described above and herein. After coupling, the modification step described herein introduces the desired modification into the bound phosphorus. The product may either go to the end of the cycle before or after deblocking 5'-OH or enter the next cycle after deblocking 5'-OH. The next cycle includes, but is not limited to, those described in Schemes I, Ib, Ic, Id, Z-1 and Z-2. It is understood by those skilled in the art that any of these may be used.

In some embodiments, the chiral reagent or salt thereof used in accordance with the present invention is of formula (Z-I).

In formula (Z-I), G ^z1 and G ^z2 are independent of each other and have a hydrogen atom, a nitro group (-NO ₂ ), a halogen atom, a cyano group (-CN), a formula (Z-II) or (Z-III). ), Or ^both G1 and G2 together form the group of formula (Z ^- IV).

、
式中、Ｇ^２１～Ｇ^２３は互いに独立して水素原子、ニトロ基、ハロゲン原子、シアノ基又はＣ_１～３アルキル基である。 In some embodiments, the basis of formula (Z-II) is as shown below:

,
In the formula, G ²¹ to G ²³ are independent of each other as a hydrogen atom, a nitro group, a halogen atom, a cyano group or a C1 _{to 3} alkyl group.

、
式中、Ｇ^３１～Ｇ^３３は互いに独立してＣ_１～４アルキル基、Ｃ_６～１４アリールＣ_１～４アルコキシ基、Ｃ_７～１４アラルキル基、Ｃ_１～４アルキルＣ_６～１４アリール基、Ｃ_１～４アルコキシＣ_６～１４アリール基、又はＣ_６～１４アリールＣ_１～４アルキル基である。 In some embodiments, the basis of formula (Z-III) is as shown below:

,
In the formula, G ³¹ to G ³³ are independent of each other, C _1-4 alkyl group, C _6-14 aryl C _1-4 alkoxy group, C _7-14 aralkyl group, C _1-4 alkyl C _6-14 aryl group. , C _{1 to 4} alkoxy C _{6 to 14} aryl groups, or C _{6 to 14} aryl C _{1 to 4} alkyl groups.

、
式中、Ｇ^４１～Ｇ^４６は互いに独立して水素原子、ニトロ基、ハロゲン原子、シアノ基又はＣ_１～３アルキル基である。 In some embodiments, the basis of formula (Z-IV) is as shown below:

,
In the formula, G ⁴¹ to G ⁴⁶ are independent of each other as a hydrogen atom, a nitro group, a halogen atom, a cyano group or a C1 _{to 3} alkyl group.

G ^z3 and G ^z4 are independent of each other and have a hydrogen atom, a C _1-3 alkyl group, a C _6-14 aryl group, or a hetero with both G ^z3 and G ^z4 together having 3 to 16 carbon atoms. An atom-containing ring is formed together with the NH component in the formula (Z—I).

、式中、Ｇ^ｚ１及びＧ^ｚ２は上に同じである。即ち、Ｇ^ｚ１及びＧ^ｚ２は、互いに独立して水素原子、ニトロ基、ハロゲン原子、シアノ基、式（Ｚ－ＩＩ）若しくは（Ｚ－ＩＩＩ）の基、又は、Ｇ^ｚ１及びＧ^ｚ２の両方が一緒になって式（Ｚ－ＩＶ）の基を形成したもの、である。 In some embodiments, the chiral reagent has the following chemical formula (Z-I'):

, In the formula, G ^z1 and G ^z2 are the same as above. That is, G ^z1 and G ^z2 are independent of each other and have a hydrogen atom, a nitro group, a halogen atom, a cyano group, a group of the formula (Z-II) or (Z-III), or both G ^z1 and G ^z2 . Together they form the group of formula (Z-IV).

In certain embodiments, the chiral reagents have a chemical formula (Z-I'), each of G ^z1 and G ^z2 is a group of formula (Z-II), in which G ²¹ -G ²³ are relative to each other. Independently, it is a hydrogen atom, a nitro group, a halogen atom, a cyano group, or a C1 _{to 3} alkyl group.

In certain embodiments, the chiral reagent has a chemical formula (Z-I'), each of G ^z1 and G ^z2 is a group of formula (Z-II), and each of G ²¹ -G ²³ is a hydrogen atom. Is.

In certain embodiments, the chiral reagents have the chemical formula (Z-I'), G ^z1 is a hydrogen atom, G ^z2 is a group of formula (Z-II), and G ²¹ -G ²³ are mutually. Independently, it is a hydrogen atom, a nitro group, a halogen atom, a cyano group or a C1 _{to 3} alkyl group.

In certain embodiments, the chiral reagent has a chemical formula (Z-I'), G ^z1 is a hydrogen atom, G ^z2 is a group of formula (Z-II), G ²¹ and G ²² respectively. Is a hydrogen atom and G ²³ is a nitro group.

In certain embodiments, the chiral reagent has a chemical formula (Z—I'), G ^z1 is a hydrogen atom, G ^z2 is a group of formula (III), and G ³¹ to G ³³ are independent of each other. C _1-4 alkyl group, C _6-14 aryl group, C _7-14 aryl group, C _1-4 alkyl C _6-14 aryl group, C _1-4 alkoxy C _6-14 aryl group, or C _{6 ~} It is a ₁₄ aryl C _1-4 alkyl group.

In some embodiments, the chiral reagent has a chemical formula (I'), G ¹ is a hydrogen atom, G ² is a group of formula (III), and G ³¹ to G ³³ are C independently of each other. _{1 to 4} alkyl group, C ₆ aryl group, C _{7 to 10} aralkyl group, C _{1 to 4} alkyl C ₆ aryl group, C _{1 to 4} alkoxy C ₆ aryl group, or C ₆ aryl C _{1 to 4} alkyl group. ..

In certain embodiments, the chiral reagents have the chemical formula (Z-I'), G ^z1 is a hydrogen atom, G ^z2 is a group of formula (Z-III), and G ³¹ -G ³³ are mutually. Independently, it is a C _1-4 alkyl group or a C ₆ aryl group. Examples of C1-4 alkyl groups include _a methyl group, an ethyl group, an n-propyl group, an iso-propyl group, an n-butyl group, and a tert-butyl group.

In certain embodiments, the chiral reagents have the chemical formula (Z-I'), G ^z1 is a hydrogen atom, G ^z2 is a group of formula (Z-III), and G ³¹ -G ³³ are mutually. Independently, it is _a C1-4 alkyl group.

In certain embodiments, the chiral reagent has a chemical formula (Z-I'), G ^z1 is a hydrogen atom, G ^z2 is a group of formula (Z-III), and G ³¹ and G ³³ are C. It is a ₆ aryl group and G ³² is a C _1-4 alkyl group.

In certain embodiments, the chiral reagent has a chemical formula (Z-I'), G ^z1 and G ^z2 together form a group of formula (Z-IV), and G ⁴¹ -G ⁴⁶ together. Independently, it is a hydrogen atom, a nitro group, a halogen atom, a cyano group, or _a C1-4 alkyl group.

In certain embodiments, the chiral reagent has the chemical formula (Z-I'), where G ^z1 and G ^z2 are combined into formula (Z-IV) (in the formula, G ⁴¹ to G ⁴⁶ are each hydrogen. Form a group (which is an atom).

の１つより選択される。 In certain embodiments, the chiral reagents are chemical formulas 3a, 3b, 5a, Z-5b, 7a, 7b, 9a, 9b, 11a, and 11b:

It is selected from one of.

、
式中、Ｇ^ｚ１～Ｇ^ｚ４は上に同じであり、Ｇ^ｚ５はヒドロキシル基の保護基であり、Ｂｓは、次の式（Ｚ－ＶＩ）～（Ｚ－ＸＩ）によって表される基より選択される基又はその誘導体である。

Ｂｓの例としては、アデニン、チミン、シトシン、グアニン、ウラシル、５－メチルシトシン、又はその誘導体があり；
Ｒ^ｚ２は互いに独立して水素、－ＯＨ、－ＳＨ、－ＮＲ^ｄＲ^ｄ、－Ｎ_３、ハロゲン、アルキル、アルケニル、アルキニル、アルキル－Ｙ^１－、アルケニル－Ｙ^１－、アルキニル－Ｙ^１－、アリール－Ｙ^１－、ヘテロアリール－Ｙ^１－、－ＯＲ^ｂ、又は－ＳＲ^ｂであり、式中、Ｒ^ｂはブロッキング成分である；
Ｙ^１はＯ、ＮＲ^ｄ、Ｓ、又はＳｅであり；
Ｒ^ｄは互いに独立して水素、アルキル、アルケニル、アルキニル、アリール、アシル、置換シリル、カルバメート、－Ｐ（Ｏ）（Ｒ^ｅ）２、又は－ＨＰ（Ｏ）（Ｒ^ｅ）であり；
Ｒ^ｅは互いに独立して水素、アルキル、アリール、アルケニル、アルキニル、アルキル－Ｙ^２－、アルケニル－Ｙ^２－、アルキニル－Ｙ^２－、アリール－Ｙ^２－、又はヘテロアリール－Ｙ^２－、又は、Ｎａ^＋、Ｌｉ^＋、若しくはＫ^＋である陽イオン、又は－Ｏ^－であり；
Ｙ^２はＯ、ＮＲ^ｄ、又はＳであり；
Ｒ^ｚ３は、－ＣＨ_２－、－（ＣＨ_２）_２－、－ＣＨ_２ＮＨ－、又は－ＣＨ_２Ｎ（ＣＨ_３）－で表される基である。 In some embodiments, the nucleoside 3'-phosphoramidite derivative used in accordance with the present invention is represented by the formula (Z-Va) or (Z-Vb):

,
In the formula, G ^z1 to G ^z4 are the same as above, G ^z5 is a protecting group for a hydroxyl group, and Bs is selected from the groups represented by the following formulas (Z-VI) to (Z-XI). A group or a derivative thereof.

Examples of Bs include adenine, thymine, cytosine, guanine, uracil, 5-methylcytosine, or derivatives thereof;
R ^z2 are independent of each other, hydrogen, -OH, -SH, -NR ^d R ^d , -N ₃ , halogen, alkyl, alkenyl, alkynyl, alkyl-Y ^1- , alkenyl-Y ^1- , alkynyl-Y ^1- . , Aryl-Y ^1- , heteroaryl-Y ^1- , -OR ^b , or -SR ^b , where R ^b is a blocking component in the formula;
Y ¹ is O, NR ^d , S, or Se;
R ^d are hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, -P (O) (R ^e ) 2, or -HP (O) (R ^e ) independently of each other;
^Re is independent of each other, hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl ^- Y2-, alkenyl ^- Y2-, alkynyl ^- Y2-, aryl ^- Y2-, or heteroaryl ^- Y2-, or , Na ⁺ , Li ⁺ , or K ⁺ cation, or ^-O- ;
Y ² is O, NR ^d , or S;
R ^z3 is a group represented by -CH ₂ -,-(CH ₂ ) _2- , -CH ₂ NH-, or -CH ₂ N (CH ₃ )-.

Examples of G5 are trityl, 4-monomethoxytrityl, 4,4'-dimethoxytrityl, 4,4', 4''-trimethoxytrityl, 9-phenylxanthine-9-yl (pixyl) and 9- ( There is p-methoxyphenyl) xanthine-9-yl (MOX).

式中、Ｇ^ｚ１、Ｇ^ｚ２、Ｇ^ｚ５、Ｂｓ、Ｒ^ｚ２、及びＲ^ｚ３のそれぞれは互いに独立して上に定義され本明細書に記載される通りである。 In some embodiments, the nucleoside 3'-phosphoramidite derivative is represented by the formula (Z-Va') or (Z-Vb'):

In the formula, G ^z1 , G ^z2 , G ^z5 , Bs, R ^z2 , and R ^z 3 are each independently defined above and as described herein.

In some embodiments, the invention relates to methods of synthesizing chiral controlled oligonucleotides.

In some embodiments, the provided method comprises reacting a molecule containing an achiral H-phosphonate component with a first activation reagent with a chiral reagent or a salt thereof to form a monomer. include. In some embodiments, the chiral reagent has the chemical formula (Z—I) and the monomer has the formula (Z—Va), (Z—Vb), (Z—Va ′), or (Z—Vb ′). Can be represented. The monomer reacts with the second activation reagent and the nucleoside to form a condensed intermediate. In some embodiments, subsequent steps involve converting the condensation intermediate into a nucleic acid containing a chiral X-phosphonate component.

In some embodiments, the methods of the invention provide a stable commercial material as a starting material. In some embodiments, the methods of the invention provide sterically controlled phosphorus atom-modified oligonucleotides with achiral starting materials.

As shown in the examples, in some embodiments, the methods of the invention do not cause degradation during the deprotection step. Furthermore, this method produces a phosphorus atom-modified oligonucleotide derivative without the need for a special capping agent.

In some embodiments, the present invention provides a method for synthesizing a sterically controlled phosphorus atom-modified oligonucleotide derivative using an achiral monomer. In some embodiments, in the first step, a nucleoside 3'-phosphoramidite derivative represented by the formula (Z-Va), (Z-Vb), (Z-Va'), or (Z-Vb'). Is reacted with a second activation reagent and a nucleoside to form a condensed intermediate. The second step converts the condensation intermediate into a nucleic acid containing a chiral X-phosphonate component.

All documents and patent applications disclosed herein are by reference to the same extent as if each individual document or patent application was specifically and individually indicated to be incorporated by reference. Incorporated in the specification.

As used in this "further embodiments of chiral reagents", the term "activating reagent" activates a less reactive site in a condensation reaction, which is attacked by a nucleophile. Refers to reagents that make it easier.

As used in this "further embodiments of chiral reagents", the "alkyl" group refers to an aliphatic hydrocarbon group. The alkyl component may be a saturated alkyl group (meaning it does not contain unsaturated units such as carbon-carbon double bonds or carbon-carbon triple bonds), or the alkyl component is an unsaturated alkyl group. May (meaning that it contains at least one unsaturated unit). The alkyl component may be branched chain, linear or cyclic moiety, whether saturated or unsaturated. The point of attachment of the alkyl is at a carbon atom that is not part of the ring. The "alkyl" component may have 1 to 10 carbon atoms (as seen herein, a numerical range such as "1 to 10" refers to each integer in a given range, eg, "1". "To 10 carbon atoms" means that the alkyl group may consist of up to 10 and a number of carbon atoms including 10 such as 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. , This definition also includes the appearance of the term "alkyl", which does not specify a numerical range). Alkyl contains both branched and linear alkyl groups. The alkyl groups of the compounds described herein may be referred to as "C ₁ to C ₆ alkyl" or similar. As just one example, "C ₁ -C ₆ alkyl" has 1, 2, 3, 4, 5, or 6 carbon atoms in the alkyl chain, i.e. the alkyl chain is, for example, methyl, ethyl, propyl, isopropyl, etc. It indicates that it is selected from n-butyl, isobutyl, sec-butyl, and tert-butyl. Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, allyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl and the like. Not limited to. In one aspect, the alkyl is a C ₁ to C ₆ alkyl. C _1-3 alkyl groups mean linear or branched alkyl groups having 1-3 carbon atoms. Examples of C _1-3 alkyl groups are methyl, ethyl, propyl and isopropyl. C _1-4 alkyl groups mean linear or branched alkyl groups with 1-4 carbon atoms. Examples of C1-4 alkyl groups include methyl _, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

As used in this "further embodiments of chiral reagents", the term "aryl" refers to an aromatic ring in which each of the atoms forming the ring is a carbon atom. Aryl rings are formed of 5, 6, 7, 8, 9, or more than 9 carbon atoms. Aryl groups are substituted or unsubstituted. In one aspect, the aryl is phenyl or naphthalenyl. Depending on the structure, the aryl group may be a monoradical or a diradical (ie, an arylene group). In one aspect, the aryl is a C ₆ to C ₁₀ aryl. C _6-14 aryl groups mean aryl groups having 6-14 carbon atoms. Examples of C _6-14 aryl groups include phenyl, biphenyl, naphthyl, anthracyl, indanyl, phthalimidyl, naphthymidyl, phenanthridinyl, and tetrahydronaphthyl.

The term "aralkyl" refers to an alkyl group substituted with an aryl group. Suitable aralkyl groups include benzyl, picoryl and the like, all of which may be substituted.

As used in this "further embodiments of chiral reagents", the "acyl component" refers to an alkyl (C = O) group, an aryl (C = O) group, or an aralkyl (C = O) group. The acyl component may have an intervening component (Y) which is oxy, amino, thio, or seleno between the carbonyl group and the hydrocarbon group. For example, the acyl group includes alkyl-Y- (C = O), aryl-Y- (C = O) or aralkyl-Y- (C = O).

As used in this "further embodiments of chiral reagents", the "alkenyl" group is a straight chain, branched chain, and cyclic hydrocarbon group containing at least one carbon-carbon double bond. The alkenyl group may be substituted.

As used in this "further embodiments of chiral reagents", the "alkynyl" group is a straight chain, branched chain, and cyclic hydrocarbon group containing at least one carbon-carbon triple bond. The alkynyl group may be substituted.

As used in this "further embodiments of chiral reagents", an "alkoxy" group refers to an oxygen-bonding alkyl group, i.e. (alkyl) -O-group, where alkyl is defined herein. It's a street. Examples include methoxy (-OCH ₃ ) or ethoxy (-OCH ₂ CH ₃ ) groups.

As used in the sections of this "further embodiments of chiral reagents", the "alkenyloxy" group refers to an alkenyl group that binds to oxygen, ie (alkenyl) -O-group, where alkenyl is defined herein. That's right.

As used in this "further embodiments of chiral reagents", an "alkynyloxy" group refers to an alkynyl group that binds to oxygen, i.e. (alkynyl) -O-group, where alkynyl is defined herein. That's right.

As used in this "further embodiments of chiral reagents", an "aryloxy" group refers to an aryl group that binds to oxygen, i.e. (aryl) -O-group, where aryl is defined herein. That's right. An example is the phenoxy (-OC ₆ H ₅ ) group.

As used in this "further embodiments of chiral reagents", the term "alkyl seleno" refers to an alkyl group to which a substituted seleno group is attached, i.e. (alkyl) -Se- group, where alkyl refers to the present. Defined in the specification.

As used in this "further embodiments of chiral reagents", the term "alkenyl seleno" refers to an alkenyl group to which a substituted seleno group is attached, i.e. (alkenyl) -Se- group, where alkenyl is the present. Defined in the specification.

As used in this "further embodiments of chiral reagents", the term "alkynyl seleno" refers to an alkynyl group with a substituted seleno group attached, i.e. (alkynyl) -Se- group, where alkenyl is the present. Defined in the specification.

As used in this "further embodiments of chiral reagents", the term "alkylthio" refers to an alkyl group attached to a crosslinked sulfur atom, i.e. (alkyl) -S-group, where alkyl is used herein. Defined. For example, alkylthio is methylthio or the like.

As used in this "further embodiments of chiral reagents", the term "alkenylthio" refers to an alkenyl group attached to a crosslinked sulfur atom, i.e. (alkenyl) -S-group, where alkenyl is herein. Defined in.

As used in this "further embodiments of chiral reagents", the term "alkynylthio" refers to an alkynyl group attached to a crosslinked sulfur atom, i.e. (alkynyl) -S-group, where alkenyl is herein. Is defined in.

As used in this "further embodiments of chiral reagents", the term "alkylamino" refers to an amino group substituted with at least one alkyl group, ie-NH (alkyl) or -N (alkyl). Refers to 2, alkyl is defined herein.

As used in this "further embodiments of chiral reagents", the term "alkenylamino" refers to an amino group substituted with at least one alkenyl group, ie-NH (alkenyl) or -N (alkenyl). Refers to 2, alkenyl as defined herein.

As used in this "further embodiments of chiral reagents", the term "alkynylamino" refers to an amino group substituted with at least one alkynyl group, ie-NH (alkynyl) or -N (alkynyl). Refers to ₂ , alkynyl is defined herein.

As used in this "further embodiments of chiral reagents", the term "halogen" is intended to include fluorine, chlorine, bromine and iodine.

As used in this "further embodiments of chiral reagents", "fluorescent group" refers to a molecule that emits light of a different wavelength when excited by light having a selective wavelength. Examples of the fluorescent group include indole group, fluorescein, tetramethylrhodamine, Texas red, body pie, 5-[(2-aminoethyl) amino] naphthalene-1-sulfonic acid (EDANS), coumarin and lucifer yellow. Not limited to.

As used in this "further embodiments of chiral reagents", the "ammonium ion" is a ^positively charged polyatomic cation of formula NH ₄₊ .

As used in this "further embodiments of chiral reagents", "alkylammonium ion" refers to an ammonium ion in which at least one of the hydrogen atoms is substituted with an alkyl group, where alkyl is defined herein. .. Examples include triethylammonium ion and N, N-diisopropylethylammonium ion.

As used in this "further embodiments of chiral reagents", the "iminium ion" has a general structure (R ^x ) ₂ C = N (R ^x ) ₂ ⁺ . The ^Rx group refers to an alkyl group, an alkenyl group, an alkynyl group, and an aryl group described herein. “Heterocyclic aromatic iminium ion” refers to an iminium ion in which nitrogen and its bonded ^Rx group form an aromatic heterocycle. "Heterocyclic iminium ion" refers to an iminium ion in which nitrogen and its bonded ^Rx groups form a heterocycle.

As used in this "further embodiments of chiral reagents", the terms "amino" or "amine" refer to -N (R ^h ) ₂ radical groups, each R ^h being referred to herein. Unless otherwise specified, hydrogen, alkyl, fluoroalkyl, carbocyclyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl are independent of each other. .. -When two N (R ^h ) groups have _two R ^h other than hydrogen, they can be combined with a nitrogen atom to form a 4, 5, 6 or 7-membered ring. For example, -N (R ^h ) ₂ is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. One or more of hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, or heteroarylalkyl are independently alkyl, heteroalkyl, alkenyl, Alkinyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilyl, -OR ⁱ , -SR ⁱ , -OC ( O) R ⁱ , -N (R ⁱ ) ₂ , -C (O) R ⁱ , -C (O) OR ⁱ , -OC (O) N (R ⁱ ) ₂ , -C (O) N (R ⁱ ) ) ₂ , -N (R ⁱ ) C (O) OR, -N (R ⁱ ) C (O) R ⁱ , -N (R ⁱ ) C (O) N (R ⁱ ) ₂ , N (R ⁱ ) C (NR ⁱ ) N (R ⁱ ) ₂ , -N (R ⁱ ) S (O) _t R ⁱ (in the formula, t is 1 or 2), -S (O), or -S (O) _t N (R ⁱ ) ₂ may be substituted with one or more substituents (where t is 1 or 2), and each ^Ri is independent of each other with hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbo. Cycrylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, or heteroarylalkyl.

As used in the section of this "further embodiments of chiral reagents", "carbamate" as used herein refers to a component of formula-C (O) OR that binds to an amino group, where R is. Alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl. Examples include Boc (tert-butyl-OC (O)-), CBz (benzyl-OC (O)-), Teoc (Me ₃ SiCH ₂ CH ₂ OC (O)-), alloc (allyl-OC (O)-). )-), Or Fmoc (9-fluorenylmethyl-OC (O)-) group, but is not limited thereto.

As used herein in the section of "further embodiments of chiral reagents", "substituted silyl" as used herein refers to a component having the formula R ^x ₃ Si-. Examples include, but are not limited to, TBDMS (tert-butyldimethylsilyl), TBDPS (tert-butyldiphenylsilyl) or TMS (trimethylsilyl).

As used in the section of this "further embodiments of chiral reagents", the term "thiol" as used herein refers to a -SH group, which comprises a substituted thiol group, i.e., a -SRJ group, where the RJ is. Independently substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclyl alkyl groups, respectively, as defined herein.

、
式中、Ｇ^ｚ１及びＧ^ｚ２は互いに独立して水素原子、ニトロ基、ハロゲン原子、シアノ基（－ＣＮ）、式（Ｚ－ＩＩ）若しくは（Ｚ－ＩＩＩ）の基、又は、Ｇ^ｚ１及びＧ^ｚ２の両方が一緒になって（Ｚ－ＩＶ）の基を形成したもの、である。いくつかの実施形態では、「キラル試薬」という用語は、立体制御されたリン原子修飾ヌクレオチド又はオリゴヌクレオチド誘導体を産生するように使用される化学組成物である。キラル試薬はヌクレオシドと反応してキラル中間体を形成する。 In some embodiments, the invention provides a chiral reagent or salt thereof. In some embodiments, the chiral reagent is of the following chemical formula (ZI):

,
In the formula, G ^z1 and G ^z2 are independent of each other, a hydrogen atom, a nitro group, a halogen atom, a cyano group (-CN), a group of the formula (Z-II) or (Z-III), or G ^z1 and G. Both of ^z2 together form the group of (Z-IV). In some embodiments, the term "chiral reagent" is a chemical composition used to produce sterically controlled phosphorus atom-modified nucleotides or oligonucleotide derivatives. Chiral reagents react with nucleosides to form chiral intermediates.

、
式中、Ｇ^２１～Ｇ^２３は互いに独立して水素原子、ニトロ基、ハロゲン原子、シアノ基、又はＣ_１～３アルキル基である。いくつかの実施形態では、Ｇ２１～Ｇ２３の例としては水素原子が挙げられる。 In some embodiments, the basis of formula (Z-II) is of the following formula:

,
In the formula, G ²¹ to G ²³ are independent of each other as a hydrogen atom, a nitro group, a halogen atom, a cyano group, or a C1 _{to 3} alkyl group. In some embodiments, examples of G21-G23 include hydrogen atoms.

、
式中、Ｇ^３１～Ｇ^３３は互いに独立してＣ_１～４アルキル基、Ｃ_６～１４アリール基、Ｃ_１～４アルコキシ基、Ｃ_７～１４アラルキル基、Ｃ_１～４アルキルＣ_６～１４アリール基、Ｃ_１～４アルコキシＣ_６～１４アリール基、又はＣ_６～１４アリールＣ_１～４アルキル基である。Ｃ_１～４アルキルＣ_６～１４アリール基の例としては、メチルフェニル基、及びエチルフェニル基が挙げられる。Ｃ_１～４アルコキシＣ_６～１４アリール基の例としては、メトキシフェニル基及びエトキシフェニル基が挙げられる。Ｃ_６～１４アリールＣ_１～４アルキル基の例としては、ベンジル基及びフェニルエチル基が挙げられる。いくつかの実施形態では、Ｇ^３１～Ｇ^３３の例としては、互いに独立してメチル基及びフェニル基であるものが挙げられる。 In some embodiments, the basis of formula (Z-III) is of the following formula:

,
In the formula, G ³¹ to G ³³ are independent of each other, C _1-4 alkyl group, C _6-14 aryl group, C _1-4 alkoxy group, C _7-14 aralkyl group, C _1-4 alkyl C _6-14 . Aryl groups, C _1-4 alkoxy C _6-14 aryl groups, or C _6-14 aryl C _1-4 alkyl groups. Examples of the C _1-4 alkyl C _6-14 aryl group include a methylphenyl group and an ethylphenyl group. C _1-4 Alkoxy C Examples of the _6-14 aryl group include a methoxyphenyl group and an ethoxyphenyl group. Examples of C _6-14 aryl C _1-4 alkyl groups include benzyl group and phenylethyl group. In some embodiments, examples of G ³¹ to G ³³ include those that are independent of each other and are methyl and phenyl groups.

、
式中、Ｇ^４１～Ｇ^４６は互いに独立して水素原子、ニトロ基、ハロゲン原子、シアノ基、又はＣ_１～３アルキル基である。いくつかの実施形態では、Ｇ４１～Ｇ４６の例としては水素原子が挙げられる。 In some embodiments, the basis of formula (Z-IV) is of the following formula:

,
In the formula, G ⁴¹ to G ⁴⁶ are independent of each other as a hydrogen atom, a nitro group, a halogen atom, a cyano group, or a C1 _{to 3} alkyl group. In some embodiments, examples of G41-G46 include hydrogen atoms.

G ^z3 and G ^z4 are independent of each other and have a hydrogen atom, a C _1-3 alkyl group, a C _6-14 aryl group, or a hetero with both G ^z3 and G ^z4 together having 3 to 16 carbon atoms. It is an atom-containing ring. ^In some embodiments, examples of ^G3 and G4 include those that together form a heteroatom-containing ring with 3-16 carbon atoms with the NH component in formula (I). ..

In certain embodiments, the chiral reagent has the following chemical formula (Z-I'):

In formula (Z-I'), G ^z1 and G ^z2 are the same above, and G ^z1 and G ^z2 are independent of each other, hydrogen atom, nitro group, halogen atom, cyano group, formula (Z-II) or The group of (Z-III), or both G ^z1 and G ^z2 are combined to form a group of formula (Z-IV).

In certain embodiments, the chiral reagents have a chemical formula (Z-I'), each of G ^z1 and G ^z2 is a group of formula (Z-II), in which G ²¹ -G ²³ are relative to each other. Independently, it is a hydrogen atom, a nitro group, a halogen atom, a cyano group, or a C1 _{to 3} alkyl group.

In certain embodiments, the chiral reagent has a chemical formula (Z-I'), each of G ^z1 and G ^z2 is a group of formula (Z-II), and each of G ²¹ -G ²³ is a hydrogen atom. Is.

In certain embodiments, the chiral reagent has a chemical formula (Z—I ′), G ^z1 is a hydrogen atom, G ^z2 is a group of formula (Z—II), and G ²¹ to G ²³ are independent of each other. It is an atom, a nitro group, a halogen atom, a cyano group, or a C1 _{to 3} alkyl group.

In certain embodiments, the chiral reagent has a chemical formula (Z-I'), G ^z1 is a hydrogen atom, G ^z2 is a group of formula (Z-II), G ²¹ and G ²² respectively. Is a hydrogen atom and G ²³ is a nitro group (-NO ₂ ).

In certain embodiments, the chiral reagents have the chemical formula (Z-I'), G ^z1 is a hydrogen atom, G ^z2 is a group of formula (Z-III), and G ³¹ -G ³³ are mutually. Independently C _1-4 alkyl group, C _6-14 aryl group, C _7-14 aryl group, C _1-4 alkyl C _6-14 aryl group, C _1-4 alkoxy C _6-14 aryl group, or C _6-14 arylC _1-4 alkyl groups.

In some embodiments, the chiral reagents have the chemical formula (Z-I'), G ^z1 is a hydrogen atom, G ^z2 is a group of formula (Z-III), and G ³¹ -G ³³ are each other. Independently, it is _{a C1-4} alkyl group or a C6 aryl group (phenyl group). Examples of the C1-4 alkyl group include _a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, and a tert-butyl group.

In certain embodiments, the chiral reagents have the chemical formula (Z-I'), G ^z1 is a hydrogen atom, G ^z2 is a group of formula (Z-III), and G ³¹ -G ³³ are mutually. Independently, it is a C _1-2 alkyl group (methyl group or ethyl group) or a _C6 aryl group (phenyl group).

In certain embodiments, the chiral reagents have the chemical formula (Z-I'), G ^z1 is a hydrogen atom, G ^z2 is a group of formula (Z-III), and G ³¹ -G ³³ are mutually. Independently, it is _a C1-4 alkyl group.

In certain embodiments, the chiral reagent has a chemical formula (Z-I'), G ^z1 is a hydrogen atom, G ^z2 is a group of formula (Z-III), and G ³¹ and G ³³ are C. It is a ₆ aryl group (phenyl group), and G ³² is a C _1-2 alkyl group.

In certain embodiments, the chiral reagent has a chemical formula (Z-I'), G ^z1 and G ^z2 together form a group of formula (Z-IV), and G ⁴¹ -G ⁴⁶ together. Independently, it is a hydrogen atom, a nitro group, a halogen atom, a cyano group, or a C1 _{to 3} alkyl group.

In certain embodiments, the chiral reagent has a chemical formula (Z—I ′) and G ^z1 and G ^z2 together form a group of formula (Z—IV), in the formula G ⁴¹ -G. Each of the ⁴⁶ is a hydrogen atom.

即ち、いくつかの実施形態では、キラル試薬は：
（Ｓ）－２－（メチルジフェニルシリル）－１－（（Ｓ）－１－ピロリジン－２－イル）エタノール（３ａ）、
（Ｒ）－２－（メチルジフェニルシリル）－１－（（Ｒ）－１－ピロリジン－２－イル）エタノール（３ｂ）、
（Ｓ）－２－（トリメチルシリル）－１－（（Ｓ）－１－ピロリジン－２－イル）エタノール（５ａ）、
（Ｒ）－２－（トリメチルシリル）－１－（（Ｒ）－１－ピロリジン－２－イル）エタノール（Ｚ－５ｂ）、
（Ｒ）－２，２－ジフェニル－１－（（Ｓ）－ピロリジン－２－イル）エタノール（７ａ）、
（Ｓ）－２，２－ジフェニル－１－（（Ｒ）－ピロリジン－２－イル）エタノール（７ｂ）、
（Ｒ）－２－（４－ニトロフェニル）－１－（（Ｓ）－ピロリジン－２－イル）エタノール（９ａ）、
（Ｓ）－２－（４－ニトロフェニル）－１－（（Ｒ）－ピロリジン－２－イル）エタノール（９ｂ）、
（Ｒ）－（９Ｈ－フルオロレン（Ｆｌｕｏｒｏｒｅｎ）－９－イル）（（Ｓ）－ピロリジン－２－イル）メタノール（１１ａ）、又は
（Ｓ）－（９Ｈ－フルオロレン－９－イル）（（Ｒ）－ピロリジン－２－イル）メタノール（１１ｂ）
より選択される。 In certain embodiments, the chiral reagent is selected from one of the chemical formulas 3a, 3b, 5a, Z-5b, 7a, 7b, 9a, 9b, 11a, and 11b:

That is, in some embodiments, the chiral reagent is:
(S) -2- (Methyldiphenylsilyl) -1-((S) -1-pyrrolidin-2-yl) ethanol (3a),
(R) -2- (Methyldiphenylsilyl) -1-((R) -1-pyrrolidin-2-yl) ethanol (3b),
(S) -2- (trimethylsilyl) -1-((S) -1-pyrrolidin-2-yl) ethanol (5a),
(R) -2- (trimethylsilyl) -1-((R) -1-pyrrolidin-2-yl) ethanol (Z-5b),
(R) -2,2-diphenyl-1-((S) -pyrrolidin-2-yl) ethanol (7a),
(S) -2,2-diphenyl-1-((R) -pyrrolidin-2-yl) ethanol (7b),
(R) -2- (4-Nitrophenyl) -1-((S) -pyrrolidin-2-yl) ethanol (9a),
(S) -2- (4-Nitrophenyl) -1-((R) -pyrrolidin-2-yl) ethanol (9b),
(R)-(9H-Fluororen-9-yl) ((S) -pyrrolidin-2-yl) methanol (11a), or (S)-(9H-fluororen-9-yl) (( R) -Pyrrolidine-2-yl) Methanol (11b)
Will be selected.

Chiral reagents react with nucleic acids or modified nucleic acids to become an asymmetry auxiliary group. The nucleoside 3'-phosphoramidite derivative is an intermediate that makes a sterically controlled phosphorus atom-modified oligonucleotide derivative, and is obtained by reacting a chiral reagent with a nucleic acid or a modified nucleic acid.

In some embodiments, the invention provides a nucleoside 3'-phosphoramidite derivative represented by the formula (Z-Va) or (Z-Vb). The compounds of formulas (Z-Va) and (Z-Vb) are known as monomers used in the synthesis of oligonucleotide derivatives. These compounds are also referred to as oxazaphosphoridine monomers. The sugar component of the compound represented by the formula (Z-Vb) is known as BNA and LNA (when R ^z3 is a methylene group).

Ｂｓの例は、アデニン、チミン、シトシン、グアニン、ウラシル、５－メチルシトシン、又はその誘導体であり；
Ｒ^ｚ２は互いに独立して水素、－ＯＨ、－ＳＨ、－ＮＲ^ｄＲ^ｄ、－Ｎ_３、ハロゲン、アルキル、アルケニル、アルキニル、アルキル－Ｙ^１－、アルケニル－Ｙ^１－、アルキニル－Ｙ^１－、アリール－Ｙ^１－、ヘテロアリール－Ｙ^１－、－ＯＲ^ｂ、又は－ＳＲ^ｂであり、式中、Ｒ^ｂはブロッキング成分であり；
Ｙ^１はＯ、ＮＲ^ｄ、Ｓ、又はＳｅであり；
Ｒ^ｄは互いに独立して水素、アルキル、アルケニル、アルキニル、アリール、アシル、置換シリル、カルバメート、－Ｐ（Ｏ）（Ｒ^ｅ）_２、又は－ＨＰ（Ｏ）（Ｒ^ｅ）であり；
Ｒ^ｅは互いに独立して水素、アルキル、アリール、アルケニル、アルキニル、アルキル－Ｙ^２－、アルケニル－Ｙ^２－、アルキニル－Ｙ^２－、アリール－Ｙ^２－、若しくはヘテロアリール－Ｙ^２－、又は、陽イオンであるＮａ^＋、Ｌｉ^＋、若しくはＫ^＋、又は－Ｏ^－であり；
Ｙ^２はＯ、ＮＲ^ｄ、又はＳであり；
Ｒ^ｚ３は－ＣＨ_２－、－（ＣＨ_２）_２－、－ＣＨ_２ＮＨ－、又は－ＣＨ_２Ｎ（ＣＨ_３）－で表される基である。 In formulas (Z-Va) and (Z-Va), G ^z1 to G ^z4 are the same above, G ^z5 is a protecting group for hydroxyl groups, and Bs is formula (Z-VI) to (Z-XI). ) Is a group selected from the groups represented by) or a derivative thereof.

Examples of Bs are adenine, thymine, cytosine, guanine, uracil, 5-methylcytosine, or derivatives thereof;
R ^z2 are independent of each other, hydrogen, -OH, -SH, -NR ^d R ^d , -N ₃ , halogen, alkyl, alkenyl, alkynyl, alkyl-Y ^1- , alkenyl-Y ^1- , alkynyl-Y ^1- . , Aryl-Y ^1- , heteroaryl-Y ^1- , -OR ^b , or -SR ^b , where R ^b is a blocking component in the formula;
Y ¹ is O, NR ^d , S, or Se;
R ^d are hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, -P (O) (R ^e ) ₂ , or -HP (O) (R ^e ) independently of each other;
^Re is independent of each other, hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl ^- Y2-, alkenyl ^- Y2-, alkynyl ^- Y2-, aryl ^- Y2-, or heteroaryl ^- Y2-, or , Cations Na ⁺ , Li ⁺ , or K ⁺ , or ^-O- ;
Y ² is O, NR ^d , or S;
R ^z3 is a group represented by -CH ₂ -,-(CH ₂ ) _2- , -CH ₂ NH-, or -CH ₂ N (CH ₃ )-.

Examples of ^Gz5 are trityl, 4-monomethoxytrityl, 4,4'-dimethoxytrityl, 4,4', 4''-trimethoxytrityl, 9-phenylxanthine-9-yl (pixil) and 9-. There is (p-methoxyphenyl) xanthine-9-yl (MOX).

、
上記式中、Ｒ^８～Ｒ^１０のそれぞれは互いに独立してＣ_１～１０アルキル、Ｃ_６～Ｃ_１０アリール、Ｃ_６～Ｃ_１０アラルキル、又はＣ_６～Ｃ_１０アリールオキシアルキルである。いくつかの実施形態では、Ｒ^８はメチル、イソプロピル、フェニル、ベンジル、及びフェノキシメチルである。いくつかの実施形態では、Ｒ^９及びＲ^１０はＣ_１～４アルキル基である。 In some embodiments, Bs is adenine, thymine, cytosine, guanine, or a derivative thereof. In some embodiments, Bs is a nucleobase or a modified nucleobase. Exemplary derivatives include, for example, those disclosed in JP2005-89441A, which are represented as follows:

,
In the above formula, each of R ⁸ to R ¹⁰ is C _{1 to 10} alkyl, C ₆ to C ₁₀ aryl, C ₆ to C ₁₀ aralkyl, or C ₆ to C ₁₀ aryl oxyalkyl independently of each other. ^In some embodiments, R8 is methyl, isopropyl, phenyl, benzyl, and phenoxymethyl. In some embodiments, R ⁹ and R ¹⁰ are C _1-4 alkyl groups.

、
式（Ｚ－Ｖａ’）及び（Ｚ－Ｖｂ’）中、Ｇ^ｚ１、Ｇ^ｚ２、Ｇ^ｚ５、Ｂｓ、Ｒ^ｚ２及びＲ^ｚ３のそれぞれは上に同じである）。ある特定の実施形態では、ヌクレオシド３’－ホスホラミダイト誘導体は、立体制御されたリン原子修飾ヌクレオチド及びオリゴヌクレオチドを産生するように用いられるキラルモノマーである。ヌクレオシド３’－ホスホラミダイト誘導体の例は、次の式で表される：
１２ａ、１２ｂ、１３ａ、１３ｂ、１４ａ、１４ｂ、１５ａ、１５ｂ、１６ａ、１６ｂ、１７ａ、１７ｂ、１８ａ、１８ｂ、１９ａ、１９ｂ、２０ａ、２０ｂ、２１ａ、２１ｂ、２２ａ、２２ｂ、２３ａ、２３ｂ、２４ａ、２４ｂ、２５ａ、２５ｂ、２６ａ、２６ｂ、２７ａ、２７ｂ、２８ａ、２８ｂ、２９ａ、２９ｂ、３０ａ、３０ｂ、３１ａ、３１ｂ、３２ａ、３２ｂ、３３ａ、３３ｂ、３４ａ、３４ｂ、及び３５ａ。

ＤＭＴｒは、４，４’－ジメトキシトリチル基を表し、ＴＯＭはトリイソプロピルシロキシメチル基を表す。 In some embodiments, the nucleoside 3'-phosphoramidite derivative is represented by the formula (Z-Va') or (Z-Vb'):

,
In formulas (Z-Va') and (Z-Vb'), G ^z1 , G ^z2 , G ^z5 , Bs, R ^z2 and R ^z3 are the same above). In certain embodiments, the nucleoside 3'-phosphoramidite derivative is a chiral monomer used to produce sterically controlled phosphorus atom-modified nucleotides and oligonucleotides. An example of a nucleoside 3'-phosphoramidite derivative is expressed by the following equation:
12a, 12b, 13a, 13b, 14a, 14b, 15a, 15b, 16a, 16b, 17a, 17b, 18a, 18b, 19a, 19b, 20a, 20b, 21a, 21b, 22a, 22b, 23a, 23b, 24a, 24b, 25a, 25b, 26a, 26b, 27a, 27b, 28a, 28b, 29a, 29b, 30a, 30b, 31a, 31b, 32a, 32b, 33a, 33b, 34a, 34b, and 35a.

DMTr represents a 4,4'-dimethoxytrityl group and TOM represents a triisopropylsiloxymethyl group.

An example using a nucleoside 3'-phosphoramidite derivative is disclosed in, for example, JP2005-89441A. By repeating the steps of condensation and deprotection, the methods of the invention facilitate chain extension of oligonucleotides, as described herein.

式（Ｚ－Ｘ）中、Ｘ^ｚはスルフィド（＝Ｓ）、Ｃ_１～３アルキル、Ｃ_１～３アルコキシ、Ｃ_１～３アルキルチオ、Ｃ_６～Ｃ_１０アリール、Ｃ_６～Ｃ_１０アラルキル、又はＣ_６～Ｃ_１０アリールオキシアルキル（ａｒｙｌｏｘｉａｌｋｙｌ）を表す。いくつかの実施形態では、Ｘ^ｚはスルフィド（＝Ｓ）を表す。ｎ^ｚは、１～１５０、１～１００、１～５０、又は１～３０を表す整数である。いくつかの実施形態では、ｎ^ｚは、好ましくは２～１００、好ましくは１０～１００、好ましくは１０～５０、及びより好ましくは１５～３０である。 In some embodiments, the oligonucleotides are as set forth in formula (ZX):

In formula (Z—X), X ^z is sulfide (= S), C _1-3 alkyl, C _1-3 alkoxy, C _1-3 alkyl thio, C ₆ to C ₁₀ aryl, C ₆ to C ₁₀ aralkyl, or Represents C ₆ to C ₁₀ aryloxyalkyl. In some embodiments, ^Xz represents sulfide (= S). n ^z is an integer representing 1 to 150, 1 to 100, 1 to 50, or 1 to 30. In some embodiments, nz is preferably ^2-100 , preferably 10-100, preferably 10-50, and more preferably 15-30.

In some embodiments, the present invention provides a method for synthesizing a sterically controlled phosphorus atom-modified oligonucleotide derivative. In some embodiments, the first step is the reaction of a molecule containing an achiral H-phosphonate component with the first activation reagent with the chiral reagent or a salt thereof to form a monomer. In some embodiments, the chiral reagent has the chemical formula (Z—I) or (Z—I ′) and the monomer has the formula (Z—Va), (Z—Vb), (Z—Va ′), or. It may be represented by (Z-Vb'). The monomer reacts with the second activation reagent and the nucleoside to form a condensed intermediate. The next step is to convert the condensation intermediate into a nucleic acid containing a chiral X-phosphonate component. In some embodiments, the method is as described in WO2010 / 064146. In some embodiments, the steps are as described in Path A and Path B of WO2010 / 064146.

In some embodiments, the invention provides a method of synthesizing a chiral controlled oligonucleotide exemplified in Scheme Z-1 below.
Scheme Z-1

Activation The achiral H-phosphonate component is treated with the first activation reagent to form the first intermediate. In one embodiment, the first activation reagent is added to the reaction mixture during the condensation step. The use of the first activation reagent depends on the reaction conditions such as the solvent used for the reaction. Examples of the first activation reagent include phosgene, trichloromethyl chlorophosphate, bis (trichloromethyl) carbonate (BTC), oxalyl chloride, Ph ₃ PCl ₂ , (PhO) ₃ PCl ₂ , N, N'-bis ( 2-oxo-3-oxazolidinyl) phosphinic acid chloride (BopCl), 1,3-dimethyl-2- (3-nitro-1,2,4-triazole-1-yl) -2-pyrrolidin-1-yl-1 , 3,2-Diazaphosphoridinium hexafluorophosphate (MNTP), or 3-nitro-1,2,4-triazole-1-yl-tris (pyrrolidin-1-yl) phosphonium hexafluorophosphate (PyNTP) be.

Examples of achiral H-phosphonate components include the compounds shown in the scheme above. DBU represents 1,8-diazabicyclo [5.4.0] undec-7-en. The H ⁺ DBU is, for example, a primary, secondary, tertiary or quaternary ammonium ion, an alkylammonium ion, a heterocyclic aromatic iminium ion, or a heterocyclic iminium ion, respectively. It may be a monovalent metal ion or it may be a monovalent metal ion.

Reaction with Chiral Reagent After the first activation step, the activated achiral H-phosphonate component reacts with the chiral reagent of formula (ZI) or (Z-I') to formulate. It forms a chiral intermediate of (Z-Va), (Z-Vb), (Z-Va'), or (Z-Vb').

Stereospecific Condensation Step The chiral intermediate of the formula Z-Va ((Z-Vb), (Z-Va'), or (Z-Vb')) is treated with a second activation reagent and a nucleoside to form a condensation intermediate. Form the body. The nucleoside may be on a solid support. Examples of the second activation reagent include 4,5-dicyanoimidazole (DCI), 4,5-dichloroimidazole, 1-phenylimidazolium triflate (PhIMT), benzimidazolium triflate (BIT), benzo. Triazole, 3-nitro-1,2,4-triazole (NT), tetrazole, 5-ethylthiotetrazole (ETT), 5-benzylthiotetrazole (BTT), 5- (4-nitrophenyl) tetrazole, Examples thereof include N-cyanomethylpyrrolidinium triflate (CMPT), N-cyanomethylpiperidinium triflate, and N-cyanomethyldimethylammonium triflate. Chiral intermediates of formula Z-Va ((Z-Vb), (Z-Va'), or (Z-Vb')) may be isolated as monomers. Normally, the chiral intermediate of Z-Va ((Z-Vb), (Z-Va'), or (Z-Vb')) is not isolated and is reacted with a nucleoside or modified nucleoside in the same container. , Chiral phosphite compounds, i.e., condensation intermediates. In another embodiment, when the method is performed by solid phase synthesis, by-products, impurities, and / or reagents are removed from the solid support containing the compound by filtration.

Capping step If the final nucleic acid is larger than the dimer, the unreacted -OH component is capped with a blocking group and the chiral auxiliary in the compound is also capped with a blocking group to form a capped condensed intermediate. You may. If the final nucleic acid is a dimer, no capping step is needed.

Modification Step The compound is modified by reaction with an electrophile. The capped condensation intermediate may be modified. In some embodiments, the modification step is performed with a sulfur electrophile, a selenium electrophile, or a boring agent. Examples of modification steps include oxidation and sulfurization steps.

In some embodiments of the method, the sulfur electrophile is a compound having one of the following formulas:
S ₈ (formula ZB), Z ^z1 -S-S-Z ^z2 , or Z ^z1 -S-V ^z -Z ^z2 ,
In the formula, Z ^z1 and Z ^z2 are independently alkyl, aminoalkyl, cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, Amides, imides, or thiocarbonyls, or Z ^z1 and Z ^z2 together to form a 3- to 8-membered alicyclic ring or heterocycle, which are substituted or substituted. It doesn't have to be;
V ^z is SO ₂ , O, or NR ^f ;
R ^f is hydrogen, alkyl, alkenyl, alkynyl, or aryl.

In some embodiments of the method, the sulfur electrophile is a compound of the following formulas ZA, ZB, ZC, ZD, ZE, or ZF:

In some embodiments, the selenium electrophile is a compound having one of the following formulas:
Se (formula Z-G), Z ^z3 -Se-Se-Z ^z4 , or Z ^z3 -Se-V ^z -Z ^z4 ,
In the formula, Z ^z3 and Z ^z4 are independently alkyl, aminoalkyl, cycloalkyl, heterocyclic, cycloalkylalkyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, Amides, imides, or thiocarbonyls, or Z ^z3 and Z ^z4 , together form a 3- to 8-membered alicyclic ring or heterocycle, which are substituted or substituted. It doesn't have to be;
V ^z is SO ₂ , S, O, or NR ^f ;
R ^f is hydrogen, alkyl, alkenyl, alkynyl, or aryl.

In some embodiments, the selenium electrophile is a compound of formula ZG, ZH, ZI, ZJ, ZK, or ZL.

In some embodiments, the borane agents are borane-N, N-diisopropylethylamine (BH ₃ DIPEA), borane-pyridine (BH ₃ Py), borane-2-chloropyridine (BH ₃ CPy), borane-aniline. (BH ₃ An), borane-tetrahydrofiyrane (BH ₃ THF), or borane-dimethylsulfide (BH ₃ Me ₂ S).

In some embodiments of the method, the modification step is an oxidation step. In some embodiments of the method, the modification step is an oxidation step with similar conditions described above in the present application. In some embodiments, the oxidation step is as disclosed, for example, in JP2010-265304A and WO2010 / 064146.

Chain Extension Cycle and Deprotection Step The capped condensation intermediate deblocks and removes the blocking group at the 5'-end of the growing nucleic acid chain to provide the compound. The compound may re-enter the chain extension cycle to form a condensation intermediate, a capped condensation intermediate, a modified and capped condensed intermediate, and a 5'-deprotected, modified and capped intermediate. good. After at least one cycle of the chain extension cycle, the 5'-deprotected, modified and capped intermediate is a chiral auxiliary ligand and other protecting groups such as nucleobases, modified nucleobases, sugars and modified sugar protecting groups. Further deblocking by removal provides nucleic acid. In another embodiment, the nucleoside containing the 5'-OH component is an intermediate from the chain extension cycle described above described herein. In yet another embodiment, the nucleoside containing the 5'-OH component is an intermediate obtained from other known nucleic acid synthesis methods. In the embodiment using a solid support, the phosphorus atom-modified nucleic acid is then cleaved from the solid support. In certain embodiments, the nucleic acid remains attached on a solid support for purification purposes and then cleaves from the solid support after purification.

In yet another embodiment, the nucleoside containing the 5'-OH component is an intermediate obtained from other known nucleic acid synthesis methods. In yet another embodiment, the nucleoside containing the 5'-OH component is an intermediate obtained from other known nucleic acid synthesis methods described herein. Still in another embodiment, the nucleoside containing the 5'-OH component is an intermediate obtained from other known nucleic acid synthesis methods comprising one or more cycles shown in Scheme I. Yet in another embodiment, the nucleoside containing the 5'-OH component is from other known nucleic acid synthesis methods comprising one or more cycles exemplified in Schemes Ib, Ic, or Id. The resulting intermediate.

In some embodiments, the present invention provides a method for synthesizing oligonucleotides using stable, commercially available materials as starting materials. In some embodiments, the invention provides a method for synthesizing oligonucleotides to produce sterically controlled phosphorus atom-modified oligonucleotide derivatives using achiral starting materials.

In some embodiments, the method does not undergo degradation under the deprotection step. Furthermore, this method produces a phosphorus atom-modified oligonucleotide derivative without the need for a special capping agent.

In some embodiments, the present invention provides a method for synthesizing a sterically controlled phosphorus atom-modified oligonucleotide derivative using an achiral monomer. In some embodiments, in the first step, a nucleoside 3'-phosphoramidite derivative represented by the formula (Z-Va), (Z-Vb), (Z-Va'), or (Z-Vb'). Is reacted with a second activation reagent and a nucleoside to form a condensed intermediate. The second step converts the condensation intermediate into a nucleic acid containing a chiral X-phosphonate component. An exemplary method is illustrated in Scheme Z-2 below.
Scheme Z-2

The detailed conditions of Scheme Z-2 are the same as those of Scheme Z-1. The starting material of formula Z-Va (Z-Vb), in particular the starting material of formula Z-Va'(or Z-Vb'), is chemically stable. As shown in the examples, this method does not cause decomposition under the deprotection step. Furthermore, this method produces a phosphorus atom-modified oligonucleotide derivative without the need for a special capping agent.

スキームＺ－３において、Ｎｕは求核試薬を表す。いくつかの実施形態では、スキームＺ－３におけるメカニズムは、前出の補助基除去メカニズムとは異なると考えられる。 In some embodiments, the mechanism of removal of auxiliary groups is as exemplified in Scheme Z-3 below.
Scheme Z-3

In Scheme Z-3, Nu represents a nucleophile. In some embodiments, the mechanism in Scheme Z-3 is considered to be different from the auxiliary group removal mechanism described above.

、
式中、Ｇ^ｚ１及びＧ^ｚ２は互いに独立して水素原子、ニトロ基、ハロゲン原子、シアノ基、式（Ｚ－ＩＩ）若しくは（Ｚ－ＩＩＩ）の基、又は、Ｇ^ｚ１及びＧ^ｚ２の両方が一緒になって式（Ｚ－ＩＶ）の基を形成したものであり、

式中、Ｇ^２１～Ｇ^２３は互いに独立して水素原子、ニトロ基、ハロゲン原子、シアノ基、又はＣ_１～３アルキル基、

であり、
式中、Ｇ^３１～Ｇ^３３は互いに独立してＣ_１～４アルキル基、Ｃ_１～４アルコキシ基、Ｃ_６～１４アリール基、Ｃ_７～１４アラルキル基、Ｃ_１～４アルキルＣ_６～１４アリール基、Ｃ_１～４アルコキシＣ_６～１４アリール基、又はＣ_６～１４アリールＣ_１～４アルキル基、

であり、
式中、Ｇ^４１～Ｇ^４６は互いに独立して水素原子、ニトロ基、ハロゲン原子、シアノ基、又はＣ_１～３アルキル基であり、Ｇ^ｚ３及びＧ^ｚ４は互いに独立して水素原子、Ｃ_１～３アルキル基、Ｃ_６～１４アリール基、又は、Ｇ^ｚ３及びＧ^ｚ４の両方が一緒になって３～１６の炭素原子を有するヘテロ原子含有環を形成したもの、である。 In some embodiments, the invention provides a chiral reagent or salt thereof having the following chemical formula (ZI):

,
In the formula, G ^z1 and G ^z2 are independent of each other and have a hydrogen atom, a nitro group, a halogen atom, a cyano group, a group of the formula (Z-II) or (Z-III), or both G ^z1 and G ^z2 . Together they formed the group of formula (Z-IV),

In the formula, G ²¹ to G ²³ are independent of each other and have a hydrogen atom, a nitro group, a halogen atom, a cyano group, or a C1 _{to 3} alkyl group.

And
In the formula, G ³¹ to G ³³ are independent of each other, C _1-4 alkyl group, C _1-4 alkoxy group, C _6-14 aryl group, C _7-14 aralkyl group, C _1-4 alkyl C _6-14 . Aryl group, C _1-4 alkoxy C _6-14 aryl group, or C _6-14 aryl C _1-4 alkyl group,

And
In the formula, G ⁴¹ to G ⁴⁶ are independent hydrogen atoms, nitro groups, halogen atoms, cyano groups, or C _1-3 alkyl groups, and G ^z 3 and G ^z 4 are independent hydrogen atoms, C ₁ It is a heteroatom-containing ring having 3 _{to 3} alkyl groups, C _{6 to 14} aryl groups, or both G ^z3 and G ^z4 together to form a heteroatom-containing ring having 3 to 16 carbon atoms.

式中、各変数は互いに独立して上に定義され本明細書に記載される通りである。いくつかの実施形態では、本発明は、式（Ｚ－１’）のキラル試薬又はその塩を提供し、Ｇ^ｚ１及びＧ^ｚ２のそれぞれは式（Ｚ－ＩＩ）の基であり、Ｇ^２１～Ｇ^２３は互いに独立して水素原子、ニトロ基、ハロゲン原子、シアノ基、又はＣ_１～３アルキル基である。いくつかの実施形態では、本発明は、式（Ｚ－１’）のキラル試薬又はその塩を提供し、Ｇ^ｚ１及びＧ^ｚ２のそれぞれは式（Ｚ－ＩＩ）の基であり、Ｇ^２１～Ｇ^２３のそれぞれは水素原子である。いくつかの実施形態では、本発明は、式（Ｚ－１’）のキラル試薬又はその塩を提供し、Ｇ^ｚ１は水素原子であり、Ｇ^ｚ２は式（Ｚ－ＩＩ）の基であり、Ｇ^２１～Ｇ^２３は互いに独立して水素原子、ニトロ基、ハロゲン原子、シアノ基、又はＣ_１～３アルキル基である。いくつかの実施形態では、本発明は、式（Ｚ－１’）のキラル試薬又はその塩を提供し、Ｇ^ｚ１は水素原子であり、Ｇ^ｚ２は式（Ｚ－ＩＩ）の基であり、Ｇ^２１及びＧ^２２のそれぞれは水素原子であり、Ｇ^２３はニトロ基である。いくつかの実施形態では、本発明は、式（Ｚ－１’）のキラル試薬又はその塩を提供し、Ｇ^ｚ１は水素原子であり、Ｇ^ｚ２は式（Ｚ－ＩＩＩ）の基であり、Ｇ^３１～Ｇ^３３は互いに独立してＣ_１～４アルキル基、Ｃ_６～１４アリール基、Ｃ_７～１４アラルキル基、Ｃ_１～４アルキルＣ_６～１４アリール基、Ｃ_１～４アルコキシＣ_６～１４アリール基、又はＣ_６～１４アリールＣ_１～４アルキル基である。いくつかの実施形態では、本発明は、式（Ｚ－１’）のキラル試薬又はその塩を提供し、Ｇ^ｚ１は水素原子であり、Ｇ^ｚ２は式（Ｚ－ＩＩＩ）の基であり、Ｇ^３１～Ｇ^３３は互いに独立してＣ_１～４アルキル基、Ｃ_６アリール基、Ｃ_７～１０アラルキル基、Ｃ_１～４アルキルＣ_６アリール基、Ｃ_１～４アルコキシＣ_６アリール基、又はＣ_６アリールＣ_１～４アルキル基である。いくつかの実施形態では、本発明は、式（Ｚ－１’）のキラル試薬又はその塩を提供し、Ｇ^ｚ１は水素原子であり、Ｇ^ｚ２は式（Ｚ－ＩＩＩ）の基であり、Ｇ^３１～Ｇ^３３は互いに独立してＣ_１～４アルキル基、又はＣ_６アリール基である。いくつかの実施形態では、本発明は、式（Ｚ－１’）のキラル試薬又はその塩を提供し、Ｇ^ｚ１は水素原子であり、Ｇ^ｚ２は式（Ｚ－ＩＩＩ）の基であり、Ｇ^３１及びＧ^３３はＣ_６アリール基であり、Ｇ^３２はＣ_１～２アルキル基である。いくつかの実施形態では、本発明は、式（Ｚ－１’）のキラル試薬又はその塩を提供し、Ｇ^ｚ１は水素原子であり、Ｇ^ｚ２は式（Ｚ－ＩＩＩ）の基であり、Ｇ^３１～Ｇ^３３は互いに独立してＣ_１～４アルキル基である。いくつかの実施形態では、本発明は、式（Ｚ－１’）のキラル試薬又はその塩を提供し、Ｇ^ｚ１は水素原子であり、Ｇ^ｚ２は式（Ｚ－ＩＩＩ）の基であり、Ｇ^３１及びＧ^３３はＣ_６アリール基であり、Ｇ^３２はＣ_１～４アルキル基である。いくつかの実施形態では、本発明は、式（Ｚ－１’）のキラル試薬又はその塩を提供し、Ｇ^ｚ１は水素原子であり、Ｇ^ｚ２は式（Ｚ－ＩＶ）の基であり、Ｇ^４１～Ｇ^４６は互いに独立して水素原子、ニトロ基、ハロゲン原子、シアノ基、又はＣ_１～３アルキル基である。いくつかの実施形態では、本発明は、式（Ｚ－１’）のキラル試薬又はその塩を提供し、Ｇ^ｚ１及びＧ^ｚ２が一緒になって式（Ｚ－ＩＶ）の基を形成し、Ｇ^４１～Ｇ^４６は互いに独立して水素原子、ニトロ基、ハロゲン原子、シアノ基、又はＣ_１～３アルキル基である。いくつかの実施形態では、本発明は、式（Ｚ－１’）のキラル試薬又はその塩を提供し、Ｇ^ｚ１及びＧ^ｚ２が一緒になって式（Ｚ－ＩＶ）の基を形成し、Ｇ^４１～Ｇ^４６のそれぞれは水素原子である。 In some embodiments, the invention provides a chiral reagent of formula Z-1 or a salt thereof having the following chemical formula (Z-I'):

In the equation, each variable is defined above independently of each other and as described herein. In some embodiments, the invention provides a chiral reagent of formula (Z-1') or a salt thereof, each of G ^z1 and G ^z2 being the group of formula (Z-II), G ^21- . G ²³ is a hydrogen atom, a nitro group, a halogen atom, a cyano group, or a C1 _{to 3} alkyl group independently of each other. In some embodiments, the invention provides a chiral reagent of formula (Z-1') or a salt thereof, each of G ^z1 and G ^z2 being the basis of formula (Z-II), G ^21- . Each of G ²³ is a hydrogen atom. In some embodiments, the invention provides a chiral reagent of formula (Z-1') or a salt thereof, where G ^z1 is a hydrogen atom and G ^z2 is a group of formula (Z-II). G ²¹ to G ²³ are independent of each other and are a hydrogen atom, a nitro group, a halogen atom, a cyano group, or a C1 _{to 3} alkyl group. In some embodiments, the invention provides a chiral reagent of formula (Z-1') or a salt thereof, where G ^z1 is a hydrogen atom and G ^z2 is a group of formula (Z-II). Each of G ²¹ and G ²² is a hydrogen atom, and G ²³ is a nitro group. In some embodiments, the invention provides a chiral reagent of formula (Z-1') or a salt thereof, where G ^z1 is a hydrogen atom and G ^z2 is a group of formula (Z-III). G ³¹ to G ³³ are independent of each other, C _1-4 alkyl group, C _6-14 aryl group, C _7-14 aralkyl group, C _1-4 alkyl C _6-14 aryl group, C _1-4 alkoxy C ₆ It is a _{to 14} aryl group or a C _{6 to 14} aryl C _{1 to 4} alkyl group. In some embodiments, the invention provides a chiral reagent of formula (Z-1') or a salt thereof, where G ^z1 is a hydrogen atom and G ^z2 is a group of formula (Z-III). G ³¹ to G ³³ are independent of each other, C _1-4 alkyl group, C ₆ aryl group, C _7-10 aralkyl group, C _1-4 alkyl C ₆ aryl group, C _1-4 alkoxy C ₆ aryl group, or C ₆ aryl C is a _{1 to 4} alkyl group. In some embodiments, the invention provides a chiral reagent of formula (Z-1') or a salt thereof, where G ^z1 is a hydrogen atom and G ^z2 is a group of formula (Z-III). G ³¹ to G ³³ are C _1-4 alkyl groups or C ₆ aryl groups independently of each other. In some embodiments, the invention provides a chiral reagent of formula (Z-1') or a salt thereof, where G ^z1 is a hydrogen atom and G ^z2 is a group of formula (Z-III). G ³¹ and G ³³ are C ₆ aryl groups, and G ³² is a C _1-2 alkyl group. In some embodiments, the invention provides a chiral reagent of formula (Z-1') or a salt thereof, where G ^z1 is a hydrogen atom and G ^z2 is a group of formula (Z-III). G ³¹ to G ³³ are C1 _{to 4} alkyl groups independently of each other. In some embodiments, the invention provides a chiral reagent of formula (Z-1') or a salt thereof, where G ^z1 is a hydrogen atom and G ^z2 is a group of formula (Z-III). G ³¹ and G ³³ are C ₆ aryl groups, and G ³² is a C _1-4 alkyl group. In some embodiments, the invention provides a chiral reagent of formula (Z-1') or a salt thereof, where G ^z1 is a hydrogen atom and G ^z2 is a group of formula (Z-IV). G ⁴¹ to G ⁴⁶ are independent hydrogen atoms, nitro groups, halogen atoms, cyano groups, or C1 _{to 3} alkyl groups. In some embodiments, the invention provides a chiral reagent of formula (Z-1') or a salt thereof, in which G ^z1 and G ^z2 together form the group of formula (Z-IV). G ⁴¹ to G ⁴⁶ are independent hydrogen atoms, nitro groups, halogen atoms, cyano groups, or C1 _{to 3} alkyl groups. In some embodiments, the invention provides a chiral reagent of formula (Z-1') or a salt thereof, in which G ^z1 and G ^z2 together form the group of formula (Z-IV). Each of G ⁴¹ to G ⁴⁶ is a hydrogen atom.

In some embodiments, the chiral reagent or salt thereof is selected from the formulas 3a, 3b, 5a, Z-5b, 7a, 7b, 9a, 9b, 11a, and 11b.

、
式中、Ｇ^ｚ１及びＧ^ｚ２は互いに独立して水素原子、ニトロ基、ハロゲン原子、シアノ基、式（Ｚ－ＩＩ）若しくは（Ｚ－ＩＩＩ）の基、又は、Ｇ^ｚ１及びＧ^ｚ２の両方が一緒になって式（Ｚ－ＩＶ）の基を形成したもの、であり、

式中、Ｇ^２１～Ｇ^２３は互いに独立して水素原子、ニトロ基、ハロゲン原子、シアノ基、又はＣ_１～３アルキル基、

であり、
式中、Ｇ^３１～Ｇ^３３は互いに独立してＣ_１～４アルキル基、Ｃ_１～４アルコキシ基、Ｃ_６～１４アリール基、Ｃ_７～１４アラルキル基、Ｃ_１～４アルキルＣ_６～１４アリール基、Ｃ_１～４アルコキシＣ_６～１４アリール基、又はＣ_６～１４アリールＣ_１～４アルキル基、

であり、
式中、Ｇ^４１～Ｇ^４６は互いに独立して水素原子、ニトロ基、ハロゲン原子、シアノ基、又はＣ_１～３アルキル基であり；
Ｇ^ｚ３及びＧ^ｚ４は互いに独立して水素原子、Ｃ_１～３アルキル基、Ｃ_６～１４アリール基、又は、Ｇ^ｚ３及びＧ^ｚ４の両方が一緒になって３～１６の炭素原子を有するヘテロ原子含有環を形成したもの、であり、
Ｇ^ｚ５はヒドロキシル基の保護基であり；
Ｒ^ｚ２は互いに独立して水素、－ＯＨ、－ＳＨ、－ＮＲ^ｄＲ^ｄ、－Ｎ_３、ハロゲン、アルキル、アルケニル、アルキニル、アルキル－Ｙ^１－、アルケニル－Ｙ^１－、アルキニル－Ｙ^１－、アリール－Ｙ^１－、ヘテロアリール－Ｙ^１－、－ＯＲ^ｂ、又は－ＳＲ^ｂであり、Ｒ^ｂはブロッキング成分であり；
Ｙ^１はＯ、ＮＲ^ｄ、Ｓ、又はＳｅであり；
Ｒ^ｄは互いに独立して水素、アルキル、アルケニル、アルキニル、アリール、アシル、置換シリル、カルバメート、－Ｐ（Ｏ）（Ｒ^ｅ）_２、又は－ＨＰ（Ｏ）（Ｒ^ｅ）であり；
Ｒ^ｅは互いに独立して水素、アルキル、アリール、アルケニル、アルキニル、アルキル－Ｙ^２－、アルケニル－Ｙ^２－、アルキニル－Ｙ^２－、アリール－Ｙ^２－、若しくはヘテロアリール－Ｙ^２－、又は、Ｎａ^＋、Ｌｉ^＋、若しくはＫ^＋である陽イオン、又は－Ｏ^－であり；
Ｙ^２はＯ、ＮＲ^ｄ、又はＳであり；
Ｒ^ｚ３は－ＣＨ_２－、－（ＣＨ_２）_２－、－ＣＨ_２ＮＨ－、又は－ＣＨ_２Ｎ（ＣＨ_３）－で表される基であり；
Ｂｓは次の式（Ｚ－ＶＩ）～（Ｚ－ＸＩ）で表される基より選択される基又はその誘導体である。

In some embodiments, the invention provides a nucleoside 3'-phosphoramidite derivative represented by the formula Z-Va or Z-Vb:

,
In the formula, G ^z1 and G ^z2 are independent of each other and have a hydrogen atom, a nitro group, a halogen atom, a cyano group, a group of the formula (Z-II) or (Z-III), or both G ^z1 and G ^z2 . Together they formed the group of formula (Z-IV),

In the formula, G ²¹ to G ²³ are independent of each other and have a hydrogen atom, a nitro group, a halogen atom, a cyano group, or a C1 _{to 3} alkyl group.

And
In the formula, G ³¹ to G ³³ are independent of each other, C _1-4 alkyl group, C _1-4 alkoxy group, C _6-14 aryl group, C _7-14 aralkyl group, C _1-4 alkyl C _6-14 . Aryl group, C _1-4 alkoxy C _6-14 aryl group, or C _6-14 aryl C _1-4 alkyl group,

And
In the formula, G ⁴¹ to G ⁴⁶ are independent hydrogen atoms, nitro groups, halogen atoms, cyano groups, or C _1-3 alkyl groups;
G ^z3 and G ^z4 are independent of each other and have a hydrogen atom, a C _1-3 alkyl group, a C _6-14 aryl group, or a hetero with both G ^z3 and G ^z4 together having 3 to 16 carbon atoms. It is an atom-containing ring.
^Gz5 is a protecting group for hydroxyl groups;
R ^z2 are independent of each other, hydrogen, -OH, -SH, -NR ^d R ^d , -N ₃ , halogen, alkyl, alkenyl, alkynyl, alkyl-Y ^1- , alkenyl-Y ^1- , alkynyl-Y ^1- . , Aryl-Y ^1- , heteroaryl-Y ^1- , -OR ^b , or -SR ^b , where R ^b is a blocking component;
Y ¹ is O, NR ^d , S, or Se;
R ^d are hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, substituted silyl, carbamate, -P (O) (R ^e ) ₂ , or -HP (O) (R ^e ) independently of each other;
^Re is independent of each other, hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl ^- Y2-, alkenyl ^- Y2-, alkynyl ^- Y2-, aryl ^- Y2-, or heteroaryl ^- Y2-, or , Na ⁺ , Li ⁺ , or K ⁺ cation, or ^-O- ;
Y ² is O, NR ^d , or S;
R ^z3 is a group represented by -CH ₂ -,-(CH ₂ ) _2- , -CH ₂ NH-, or -CH ₂ N (CH ₃ )-;
Bs is a group or a derivative thereof selected from the groups represented by the following formulas (Z-VI) to (Z-XI).

、
式中、各変数は互いに独立して上に定義され本明細書に記載される通りである。 In some embodiments, the invention provides a nucleoside 3'-phosphoramidite derivative of formula Z-Va or Z-Vb having a structure of (Z-Va') or (Z-Vb'):

,
In the equation, each variable is defined above independently of each other and as described herein.

In some embodiments, the present invention relates to formulas 12a, 12b, 13a, 13b, 14a, 14b, 15a, 15b, 16a, 16b, 17a, 17b, 18a, 18b, 19a, 19b, 20a, 20b, 21a, 21b, 22a, 22b, 23a, 23b, 24a, 24b, 25a, 25b, 26a, 26b, 27a, 27b, 28a, 28b, 29a, 29b, 30a, 30b, 31a, 31b, 32a, 32b, 33a, 33b, A nucleoside 3'-phosphoramidite derivative selected from 34a, 34b, and 35a is provided. In some embodiments, the present invention relates to formulas 12a, 12b, 13a, 13b, 14a, 14b, 15a, 15b, 16a, 16b, 17a, 17b, 18a, 18b, 19a, 19b, 20a, 20b, 21a, Provided is a nucleoside 3'-phosphoramidite derivative selected from 21b, 22a, 22b, 23a, 23b, 24a, 24b, 25a, 25b, 26a, or 26b.

In some embodiments, the present invention provides a method for synthesizing a sterically controlled phosphorus atom-modified oligonucleotide derivative.
With the step of reacting a molecule containing an achiral H-phosphonate component, a chiral reagent or a salt thereof to form a monomer of a nucleoside 3'-phosphoramidite derivative;
With the step of reacting the monomer and the nucleoside to form a condensed intermediate;
Containing a step of converting the condensation intermediate into a nucleic acid containing a chiral X-phosphonate component.
The chiral reagent has the following chemical formula (Z-I).

In some embodiments, the present invention provides a method for synthesizing a sterically controlled phosphorus atom-modified oligonucleotide derivative.
With the step of reacting a molecule containing an achiral H-phosphonate component, a chiral reagent or a salt thereof to form a monomer of a nucleoside 3'-phosphoramidite derivative;
With the step of reacting the monomer and the nucleoside to form a condensed intermediate;
Containing a step of converting the condensation intermediate into a nucleic acid containing a chiral X-phosphonate component.
The chiral reagent has the following chemical formula (Z-I').

In some embodiments, the present invention provides a method for synthesizing a sterically controlled phosphorus atom-modified oligonucleotide derivative.
With the step of reacting a molecule containing an achiral H-phosphonate component, a chiral reagent or a salt thereof to form a monomer of a nucleoside 3'-phosphoramidite derivative;
With the step of reacting the monomer and the nucleoside to form a condensed intermediate;
Containing a step of converting the condensation intermediate into a nucleic acid containing a chiral X-phosphonate component.
The chiral reagent is selected from the formulas 3a, 3b, 5a, Z-5b, 7a, 7b, 9a, 9b, 11a and 11b.

In some embodiments, the present invention provides a method for synthesizing a sterically controlled phosphorus atom-modified oligonucleotide derivative.
A step of reacting a nucleoside 3'-phosphoramidite derivative represented by the formula (Z-Va) or (Z-Vb) with an activation reagent and a nucleoside to form a condensed intermediate;
The step of converting the condensation intermediate into a nucleic acid containing a chiral X-phosphonate component,
including.

In some embodiments, the present invention provides a method for synthesizing a sterically controlled phosphorus atom-modified oligonucleotide derivative.
A step of reacting a nucleoside 3'-phosphoramidite derivative represented by the formula (Z-Va) or (Z-Vb) with an activation reagent and a nucleoside to form a condensed intermediate;
Includes the step of converting the condensation intermediate into a nucleic acid containing a chiral X-phosphonate component;
The nucleoside 3'-phosphoramidite derivative represented by the formula (Z-Va) or (Z-Vb) is represented by the formulas 12a, 12b, 13a, 13b, 14a, 14b, 15a, 15b, 16a, 16b, 17a, 17b, 18a. , 18b, 19a, 19b, 20a, 20b, 21a, 21b, 22a, 22b, 23a, 23b, 24a, 24b, 25a, 25b, 26a, 26b, 27a, 27b, 28a, 28b, 29a, 29b, 30a, 30b. , 31a, 31b, 32a, 32b, 33a, 33b, 34a, 34b, and 35a.
In some embodiments, the present invention provides a method for synthesizing a sterically controlled phosphorus atom-modified oligonucleotide derivative.
A step of reacting a nucleoside 3'-phosphoramidite derivative represented by the formula (Z-Va) or (Z-Vb) with an activation reagent and a nucleoside to form a condensed intermediate;
Includes the step of converting the condensation intermediate into a nucleic acid containing a chiral X-phosphonate component;
The nucleoside 3'-phosphoramidite derivative represented by the formula (Z-Va) or (Z-Vb) is represented by the formulas 12a, 12b, 13a, 13b, 14a, 14b, 15a, 15b, 16a, 16b, 17a, 17b, 18a. , 18b, 19a, 19b, 20a, 20b, 21a, 21b, 22a, 22b, 23a, 23b, 24a, 24b, 25a, 25b, 26a, and 26b.

Preparation and Use of Predetermined Chiral Auxiliaries of Formula Z-I
Abbreviation
ac: Acetyl bz: Benzoyl CSO: (1S)-(+)-(10-camphorsulfonyl) Oxaziridine DBU: 1,8-diazabicyclo [5.4.0] Undec-7-endCA: Dichloroacetic acid DCM : Dichloromethane, CH ₂ Cl ₂
Tr: Trityl, TriphenylmethylMeIm: N-Methylimidazole NIS: N-iodosuccinimide pac: Phenoxyacetyl Ph: Phenyl PhIMT: N-Phenylimidazolium triflate POS: 3-Phenyl-1,2,4-dithiazoline ) -5-on TBS: tert-butyldimethylsilyl TBDPS: tert-butyldiphenylsilyl TOM: triisopropylsiloxymethyl TFA: trifluoroacetic acid

General procedure for the synthesis of chiral controlled oligonucleotides-1
Automated solid phase synthesis of chiral controlled oligonucleotides was performed according to the cycle shown in Table Z-1.

Table Z-1 Synthesis procedure

General procedure for the synthesis of chiral controlled oligonucleotides-2
Automated solid phase synthesis of chiral controlled oligonucleotides was performed according to the cycle shown in Table Z-2.

Table Z-2

^* Preparation of pre-activated monomer in step 2 of Table Z-2 The nucleoside-3'-H-phosphonate monoester is dehydrated by repeated azeotropic boiling with dehydrated toluene, and then dissolved in the dehydrated MeCN. Ph ₃ PCl ₂ is added to the solution and the mixture is stirred for 5 minutes. Azeotrope is repeated with dehydrated toluene, a solution of the chiral reagent dissolved in the dehydrated MeCN is added dropwise to the mixture with a syringe, and the mixture is stirred under argon for 5 minutes.

After synthesis, the resin was treated with 25% NH ₃ aqueous solution (1 mL) at 55 ° C. for 12 hours. The mixture was cooled to room temperature and the resin was removed by membrane filtration. The filtrate was concentrated to dryness under vacuum. The residue is dissolved in _H2O (3 mL) and by RP-UPLC-MS at 50 ° C. with a linear gradient of acetonitrile (0-50% / 30 min) in 0.1 M triethylammonium acetate buffer (pH 7.0). Below, analysis was performed at a rate of 0.3 mL / min.

Example Z-1

(S) -1-tritylpyrrolidine-2-carbaldehyde (1a)

Compound 1a was synthesized from L-proline according to the procedure described in the literature (Guga, P. Curr. Top. Med. Chem. 2007, 7, 695-713.).

(R) -1-tritylpyrrolidine-2-carbaldehyde (1b)

Compound 1b was synthesized from D-proline in the same manner as compound 1a.

(S) -2- (Methyldiphenylsilyl) -1-((S) -1-tritylpyrrolidine-2-yl) ethanol (2a)

THF (30 mL) in a solution of methyldiphenylsilylmethyl magnesium chloride in THF prepared from chloromethyldiphenylmethylsilane (4.02 g, 16.3 mmol) in THF (14 mL) and magnesium (402 mg, 16.3 mmol) in THF. ) 1a (2.79 g, 8.14 mmol) solution was added. After stirring on ice for 1.5 hours, the mixture was warmed to room temperature and stirring was continued for 30 minutes. Saturated aqueous NH ₄ Cl (100 mL) was added to the reaction mixture at 0 ° C. and extraction was performed 3 times with diethyl ether (100 mL). The extracts were combined, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. When the residue was chromatographed on silica gel, 2a was obtained as a colorless foam (3.91 g, 87%).
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.48-7.08 (25H, m), 4.33-4-23 (1H, m), 3.16-2.89 (3H, m), 2. 84 (1H, brass), 1.70-1.54 (1H, m), 1.35 (1H, dd, J = 14.7, 6.3Hz), 1.10 (1H, dd, J = 14) .7, 8.1Hz), 1.18-1.05 (1H, m), 1.04-0.90 (1H, m), 0.34 (3H, s), -0.17--0 .36 (1H, m).

(S) -2- (Methyldiphenylsilyl) -1-((S) -1-pyrrolidin-2-yl) ethanol (3a)

2a (3.91 g, 7.06 mmol) was dissolved in 3% DCA in DCM (70 mL) and stirred at room temperature for 10 minutes. 1M NaOH (200 mL) was added to the mixture and extraction was performed 3 times with DCM (100 mL). The extracts were combined, dried over Na ₂ SO, filtered and concentrated under reduced pressure. Chromatographing the residue on silica gel gave 3a as a pale yellow oil (1.99 g, 90%).
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.57-7.52 (5H, m), 7.38-7.33 (5H, m), 3.77 (1H, ddd, J = 8.9,5) .4,3.5Hz), 3.01 (1H, dt, J = 7.4,3.6Hz), 2.97-2.79 (2H, m), 2.27 (2H, brass), 1 .76-1.53 (4H, m), 1.38 (1H, dd, J = 15.0, 9.0Hz), 1.24 (1H, dd, J = 15.0, 5.4Hz), 0.65 (3H, s); ¹³ C NMR (100.4 MHz, CDCl ₃ ) δ137.4, 137.1,134.6,134.5, 129.1, 127.8, 69.5, 64. 1,47.0, 25.8, 24.0, 19.6, -3.4. MALDIOF-MS m / z C ₁₉ H ₂₆ NOSi [M + H] ⁺ calculated value 312.18, measured value 312.06.

Example Z-2

(R) -2- (Methyldiphenylsilyl) -1-((R) -1-tritylpyrrolidine-2-yl) ethanol (2b)

Compound 2b was obtained in the same manner as compound 2a, but with 1b instead of 1a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.48-7.12 (25H, m), 4.33-4.24 (1H, m), 3.16-2.89 (3H, m), 2. 86 (1H, brass), 1.69-1.52 (1H, m), 1.35 (1H, dd, J = 14.4, 6.0Hz), 1.10 (1H, dd, J = 14) .4,8.4Hz), 1.18-1.05 (1H, m), 1.03-0.89 (1H, m), 0.33 (3H, s), -0.19--0 .39 (1H, m); ¹³ C NMR (75.5 MHz, CDCl ₃ ) δ144.5, 137.5, 136.8, 134.6, 134.3, 129.8, 129.0, 127.8 , 127.7, 127.4, 126.1, 77.9, 71.7, 65.1, 53.5, 25.0, 24.8, 19.6, -4.0. MALDIOF-MS m / z C ₃₈ H ₄₀ NOSi [M + H] ⁺ calculated value 554.29, measured value 554.09.

(R) -2- (Methyldiphenylsilyl) -1-((R) -1-pyrrolidin-2-yl) ethanol (3b)

Compound 3b was obtained in the same manner as compound 3a, but with 2b instead of 2a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.58-7.52 (5H, m), 7.38-7.33 (5H, m), 3.78 (1H, ddd, J = 9.0, 5) .1,3.6Hz), 3.00 (1H, dt, J = 7.4,3.3Hz), 2.97-2.78 (2H, m), 2.19 (2H, brass), 1 .76-1.53 (4H, m), 1.38 (1H, dd, J = 14.6, 9.0Hz), 1.24 (1H, dd, J = 14.6, 5.1Hz), 0.66 (3H, s); ¹³ C NMR (75.5 MHz, CDCl ₃ ) δ137.5, 137.1,134.5, 134.4, 129.0, 127.7, 69.2, 64. 2,46.9,25.8,24.0,19.7,-3.4. MALDIOF-MS m / z C ₁₉ H ₂₆ NOSi [M + H] ⁺ calculated value 312.18, measured value 312.09.

Example Z-3

(S) -2- (trimethylsilyl) -1-((S) -1-tritylpyrrolidine-2-yl) ethanol (4a)

Compound 4a was obtained in the same manner as compound 2a, but with "chloromethyltrimethylsilane" instead of "chloromethyldiphenylmethylsilane".
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.58-7.51 (5H, m), 7.31-7.14 (10H, m), 4.13 (1H, dt, J = 7.5, 3) .0Hz), 3.39-3.31 (1H, m), 3.20-2.99 (2H, m), 2.84 (1H, s), 1.74-1.57 (1H, m) ), 1.29-1.10 (2H, m), 0.74 (1H, dd, J = 14.4,7.2Hz), 0.46 (1H, dd, J = 14.4,7. 2Hz), -0.15 (9H, s). MALDITF-MS m / z C ₂₈ H ₃₆ NOSi [M + H] ⁺ calculated value 430.26, measured value 430.09.

(S) -2- (trimethylsilyl) -1-((S) -1-pyrrolidin-2-yl) ethanol (5a)

Compound 5a was obtained in the same manner as compound 3a, but with 4a instead of 2a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ3.76 (1H, ddd, J = 8.8, 5.7, 3.3 Hz), 3.08 (1 H, dt, J = 7.8, 3.3 Hz) , 3.02-2.87 (2H, m), 2.48 (2H, brs), 1.81-1.58 (4H, m), 0.83 (1H, dd, J = 14.7, 8.7Hz), 0.68 (1H, dd, J = 14.7, 6.0Hz), 0.05 (9H, s); ¹³ C NMR (75.5MHz, CDCl ₃ ) δ69,6,64. 3,46.9, 25.8, 23.9, 22.0, -0.8. MALDIOF-MS m / z C ₉ H ₂₂ NOSi [M + H] ⁺ calculated value 188.15, measured value 188.00.

Example Z-5

(R) -2,2-diphenyl-1-((S) -1-tritylpyrrolidine-2-yl) ethanol (6a)

N-BuLi (1.67 M solution of hexane, 24 mL, 40 mmol) was added dropwise to a solution of diphenylmethane (6.7 mL, 40 mmol) in anhydrous THF (36 mL) at room temperature, and the mixture was stirred for 1 hour. 1a (3.41 g, 10 mmol) dehydrated by repeated azeotropic distillation with toluene in anhydrous THF (40 mL) was slowly added to the mixture at 0 ° C. and stirring was continued for 45 minutes. Then, saturated NH ₄ Cl aqueous solution (100 mL) and Et ₂ O (100 mL) were added, the organic layer was separated, and the aqueous layer was extracted with Et ₂ O (2 × 100 mL). The organic layers were combined, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by chromatography on silica gel to give 6a (1.41 g, 28%) as a white foam.

(R) -2,2-diphenyl-1-((S) -pyrrolidin-2-yl) ethanol (7a)

6a (650 mg, 1.27 mmol) was dissolved in 3% DCA in DCM (13 mL) and stirred at room temperature for 10 minutes. 1M NaOH (40 mL) was added to the mixture and extraction was performed 3 times with DCM (30 mL). The extracts were combined, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Chromatography of the residue on silica gel gave 7a as a pale yellow oil (316 mg, 93%).
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.44-7.38 (2H, m), 3.33-7.14 (8H, m), 4.46 (1H, dd, J = 9.9, 3) .3Hz), 3.91 (1H, d, J = 9.9Hz), 3.02-2.88 (2H, m), 2.81-2.69 (1H, m), 2.52 (2H) , Brs), 1.88-1.56 (4H, m); ¹³ C NMR (75.5 MHz, CDCl ₃ ) δ142.3, 142.0, 128.6, 128.5, 128.4, 128. 2,126.5,126.4,73.5,60.1,55.8,46.6,25.8,23.4. MALDIOF-MS m / z C ₁₈ H ₂₂ NO [M + H] ⁺ calculated value 268.17, measured value 268.06.

Example Z-6

(S) -2,2-diphenyl-1-((R) -1-tritylpyrrolidine-2-yl) ethanol (6b)

Compound 6b was obtained in the same manner as compound 6a, but with 1b instead of 1a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.44-7.37 (6H, m), 7.30-7.01 (17H, m), 6.66-6.61 (2H, m), 4. 80 (1H, d, J = 10.8Hz), 3.63 (1H, d, J = 10.8Hz), 3.36-3.28 (1H, m), 3.22-3.09 (1H) , M), 3.01-2.89 (1H, m), 2.66 (1H, s), 1.90-1.75 (1H, m), 1.29-1.04 (2H, m) ), 0.00-0.19 (1H, m); ¹³ C NMR (75.5 MHz, CDCl ₃ ) δ144.2, 142.9, 141.6, 130.0, 128.5, 128.4 , 127.9, 127.8, 127.4, 126.4, 126.2, 77.9, 75.9, 61.9, 55.4, 53.4, 24.7, 24.5. MALDIOF-MS m / z C ₃₇ H ₃₆ NO [M + H] ⁺ calculated value 510.28, measured value 510.11.

(S) -2,2-diphenyl-1-((R) -pyrrolidin-2-yl) ethanol (7b)

Compound 7b was obtained in the same manner as compound 7a, but with 6b instead of 6a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.45-7.14 (10H, m), 4.45 (1H, dd, J = 9.9, 3.3 Hz), 3.91 (1H, d, J) = 9.9Hz), 3.00-2.89 (2H, m), 2.82-2.71 (1H, m), 2.40 (2H, brass), 1.87-1.55 (4H) , M); ¹³ C NMR (75.5 MHz, CDCl ₃ ) δ142.3, 142.0, 128.5, 128.3, 128.1, 126.3, 126.2, 73.4, 60.1 , 55.9, 46.5, 25.8, 23.5. MALDIOF-MS m / z C ₁₈ H ₂₂ NO [M + H] ⁺ calculated value 268.17, measured value 268.03.

Example Z-7

(R) -2- (4-Nitrophenyl) -1-((S) -1-tritylpyrrolidine-2-yl) ethanol (8a)

Compound 8a was obtained in the same manner as compound 6a, but with "4-nitrobenzyl chloride" instead of "diphenylmethane".
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.09-8.03 (2H, m), 7.49-7.43 (6H, m), 7.28-7.09 (11H, m), 4. 23 (1H, ddd, J = 8.3,5.6,3.0Hz), 3.43-3.33 (1H, m), 3.23-3.11 (1H, m), 3.07 -2.96 (1H, m), 2.83 (1H, brass), 2.74 (1H, dd, J = 13.8, 8.4Hz), 2.49 (1H, dd, J = 13. 8,5.1 Hz), 1.83-1.67 (1H, m), 1.41-1.17 (2H, m), 0.27-0.08 (1H, m); ¹³ C NMR ( 75.5MHz, CDCl ₃ ) δ147.3, 146.3, 144.3, 129.8, 129.6, 127.5, 126.3, 123.4, 77.9, 74.8, 63.5 , 53.2, 39.5, 25.0, 24.9. MALDITF-MS m / z C ₃₁ H ₃₁ N ₂ O ₃ [M + H] ⁺ calculated value 479.23, measured value 479.08.

(R) -2- (4-Nitrophenyl) -1-((S) -pyrrolidin-2-yl) ethanol (9a)

Compound 9a was obtained in the same manner as compound 7a, but with 8a instead of 6a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.15 (2H, d, J = 8.7 Hz), 7.42 (2H, d, J = 8.7 Hz), 3.86-3.79 (1H, m) ), 3.16-3.07 (1H, m), 2.99-2.68 (6H, m), 1.84-1.68 (4H, m); ¹³ C NMR (75.5 MHz, CDCl) ₃ ) δ147.4, 146.2, 129.9, 123.2, 72.4, 62.0, 46.6, 40.4, 25.7, 24.4. MALDITF-MS m / z C ₁₂ H ₁₇ N ₂ O ₃ [M + H] ⁺ calculated value 237.12, measured value 237.01.

Example Z-8

(S) -2- (4-Nitrophenyl) -1-((R) -1-tritylpyrrolidine-2-yl) ethanol (8b)

Compound 8b was obtained in the same manner as compound 8a, but with 1b instead of 1a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.09-8.04 (2H, m), 7.49-7.43 (6H, m), 7.28-7.09 (11H, m), 4. 22 (1H, ddd, J = 8.4,5.6,3.0Hz), 3.43-3.33 (1H, m), 3.24-3.10 (1H, m), 3.08 -2.94 (1H, m), 2.81 (1H, brass), 2.75 (1H, dd, J = 14.0, 8.1Hz), 2.49 (1H, dd, J = 14. 0,5.1 Hz), 1.81-1.67 (1H, m), 1.40-1.16 (2H, m), 0.26-0.09 (1H, m); ¹³ C NMR ( 75.5MHz, CDCl ₃ ) δ147.3, 144.3, 129.8, 129.6, 129.4, 126.3, 123.5, 77.9, 74.8, 63.5, 53.2 , 39.5, 25.0, 24.9. MALDITF-MS m / z C ₃₁ H ₃₁ N ₂ O ₃ [M + H] ⁺ calculated value 479.23, measured value 479.08.

(S) -2- (4-Nitrophenyl) -1-((R) -pyrrolidin-2-yl) ethanol (9b)

Compound 9b was obtained in the same manner as compound 9a, but with 8b instead of 8a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.19-8.13 (2H, m), 7.45-7.39 (2H, m), 3.83 (1H, ddd, J = 7.7, 5) .4, 3.9Hz), 3.14 (1H, dt, J = 7.7, 3.9Hz), 3.01-2.87 (2H, m), 2.83 (1H, d, J =) 3.3Hz), 2.81 (1H, s), 2.62 (2H, brass), 1.79-1.72 (4H, m); ¹³ C NMR (75.5MHz, CDCl ₃ ) δ147.3 , 146.5, 130.0, 123.5, 72.7, 61.7, 46.7, 40.1, 25.8, 24.2. MALDITF-MS m / z C ₁₂ H ₁₇ N ₂ O ₃ [M + H] ⁺ calculated value 237.12, measured value 237.02.

Example Z-9

(R)-(9H-Fluorene-9-yl) ((S) -1-tritylpyrrolidine-2-yl) Methanol (10a)

Compound 10a was obtained in the same manner as compound 6a, but with "fluorene" instead of "diphenylmethane".
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.70 (1H, d, J = 7.5 Hz), 7.66 (1H, d, J = 7.8 Hz), 7.55 (2H, d, J = 7) .5Hz), 7.44-7.09 (18H, m), 6.87-6.62 (1H, m), 4.55-4.48 (1H, m), 4.06 (1H, d) , J = 7.5Hz), 3.43-3.34 (1H, m), 3.18-3.06 (1H, m), 2.98-2.88 (1H, m), 2.85 (1H, brass), 1.42-1.24 (1H, m), 1.18-1.04 (1H, m), 0.53-0.39 (1H, m), -0.02- -0.20 (1H, m); MALDITOF-MS m / z C ₃₇ H ₃₄ NO [M + H] ⁺ calculated value 508.26, measured value 508.12.

(R)-(9H-fluororen-9-yl) ((S) -pyrrolidin-2-yl) methanol (11a)

Compound 11a was obtained in the same manner as compound 7a, but with 10a instead of 6a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.76 (2H, d, J = 7.5 Hz), 7.68 (2H, t, J = 8.0 Hz), 7.43-7.35 (2H, m) ), 7.34-7.25 (2H, m), 4.28 (1H, d, J = 6.3Hz), 4.03 (1H, dd, J = 6.5, 4.2Hz), 3 .19-3.11 (1H, m), 2.97-2.88 (1H, m), 2.86-2.76 (1H, m), 2.02 (2H, brs), 1.77 -1.53 (3H, m), 1.38-1.23 (1H, m); MALDITOF-MS m / z C ₁₈ H ₂₀ NO [M + H] ⁺ calculated value 266.15, measured value 266.04 ..

(S) -2-tosyl-1-((S) -1-tritylpyrrolidine-2-yl) ethanol (12a')

Compound 12a'was obtained in the same manner as compound 2a, but with "chloromethyl p-tolyl sulfone" instead of "chloromethyl diphenylmethyl silane".

^{1 1} H NMR (600 MHz, CDCl ₃ ) δ7.66 (2H, d, J = 8.4 Hz), 7.48-7.44 (6H, m), 7.35 (2H, d, J = 7.2 Hz) ), 7.21-7.13 (9H, m), 4.39-4.36 (1H, m), 3.33 (1H, s), 3.24-3.20 (1H, m), 3.19-3.10 (2H, m), 2.98-2.92 (2H, m), 2.49 (3H, s), 1.55-1.49 (1H, m), 1. 33-1.26 (1H, m), 1.12-1.04 (1H, m), 0.22-0.14 (1H, m); ¹³ C NMR (150.9 MHz, CDCl ₃ ) δ144. 6,144.5, 136.3, 129.9, 129.5, 128.1, 127.5, 126.2, 78.0, 69.1, 63.9, 60.2, 52.6 25.5, 24.7, 21.7.

(S) -2-tosyl-1-((S) -1-tritylpyrrolidine-2-yl) ethanol (13a')

Compound 13a'was obtained in the same manner as compound 3a, but with 12a'instead of 2a.

^{1 1} H NMR (600 MHz, CDCl ₃ ) δ7.82 (2H, d, J = 8.4 Hz), 7.37 (2H, d, J = 8.4 Hz), 4.01 (1H, ddd, J = 12) .0, 5.1, 3.0Hz), 3.32 (1H, dd, J = 14.4,3.0Hz), 3.25 (1H, dd, J = 14.4,9.0Hz), 3.16 (1H, dt, J = 7.8, 5.1Hz), 2.90-2.82 (2H, m), 2.46 (3H, s), 2.04 (2H, brs), 1.78-1.63 (3H, m), 1.62-1.55 (1H, m); ¹³ C NMR (150.9MHz, CDCl ₃ ) δ144.5, 136.7, 129.7, 127 .7, 67.4, 61.8, 60.1, 46.7, 25.7, 21.4. MALDITF-MS m / z C ₁₃ H ₂₀ NO ₃ S [M + H] ⁺ calculated value 270.12, measured value 270.04.

(R) -2-tosyl-1-((R) -1-tritylpyrrolidine-2-yl) ethanol (12b')

Compound 12b'was obtained using 1b instead of 1a in the same manner as compound 12a'.

^{1 1} H NMR (600 MHz, CDCl ₃ ) δ7.66 (2H, d, J = 8.4 Hz), 7.47-7.44 (6H, m), 7.35 (2H, d, J = 7.8 Hz) ), 7.21-7.13 (9H, m), 4.37 (1H, dt, J = 8.6, 2.4Hz), 3.33 (1H, s), 3.23-3.20 (1H, m), 3.19-3.12 (2H, m), 2.98-2.92 (2H, m), 2.49 (3H, s), 1.56-1.49 (1H) , M), 1.32-1.26 (1H, m), 1.11-1.03 (1H, m), 0.23-0.15 (1H, m); ¹³ C NMR (150.9 MHz) , CDCl ₃ ) δ144.6, 144.5, 136.3, 129.9, 129.6, 128.1, 127.6, 126.2, 78.0, 69.1, 63.9, 60. 2,52.6, 25.5, 24.7, 21.7.

(R) -2-tosyl-1-((R) -1-tritylpyrrolidine-2-yl) ethanol (13b')

Compound 13b'was obtained in the same manner as compound 13a', but with 12b'instead of 12a'.

^{1 1} H NMR (600 MHz, CDCl ₃ ) δ7.82 (2H, d, J = 8.4 Hz), 7.37 (2H, d, J = 8.4 Hz), 4.01 (1H, ddd, J = 9) .0,5.1,3.0Hz), 3.32 (1H, dd, J = 14.4,3.0Hz), 3.25 (1H, dd, J = 14.4,9.0Hz), 3.17 (1H, dt, J = 7.2, 5.1Hz), 2.89-2.83 (2H, m), 2.46 (3H, s), 2.04 (2H, brs), 1.79-1.64 (3H, m), 1.62-1.55 (1H, m); ¹³ C NMR (150.9MHz, CDCl ₃ ) δ144.8, 136.6, 129.8,127 9.9, 67.7, 61.8, 60.1, 46.8, 25.9, 25.8, 21.6. MALDITF-MS m / z C ₁₃ H ₂₀ NO ₃ S [M + H] ⁺ calculated value 270.12, measured value 270.05.

Example Z-10

Oxazaphosphorysine monomer 12a

3a (560 mg, 1.80 mmol) was dehydrated by repeated azeotropic boiling with dehydrated toluene and dissolved in dehydrated diethyl ether (0.90 mL) under argon. N-Methylmorpholine (400 microL, 3.60 mmol) was added to the solution, and the resulting solution was added to a solution of PCL ₃ (160 microL, 1.80 mmol) in dehydrated diethyl ether (0.90 mL) under argon. The mixture was added dropwise at 0 ° C. with stirring. The mixture was then warmed to room temperature and stirred for 30 minutes. The obtained N-methylmorpholine hydrochloride was removed by filtration through nitrogen, and the filtrate was concentrated to dryness under reduced pressure to give a crude 2-chloro-1,3,2-oxazaphosphoridine derivative. The crude material was dissolved in freshly distilled THF (3.6 mL) to make a 0.5 M solution, which was used to synthesize the nucleoside 3'-O-oxazaphosphoridine without further purification. ..

5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine (636 mg, 0.84 mmol) is dehydrated by repeated azeotropic boiling with dehydrated toluene and under argon. , Dissolved in freshly distilled THF (2.5 mL). Et ₃ N (0.58 mL, 4.2 mmol) was added and the mixture was cooled to −78 ° C. A 0.5 M solution of the crude 2-chloro-1,3,2-oxazaphosphoridine derivative in freshly distilled THF (3.6 mL, 1.80 mmol) is added dropwise with a syringe and the mixture is added at room temperature to 15 Stir for minutes. Then, saturated NaHCO ₃ aqueous solution (70 mL) and CHCl ₃ (70 mL) were added, the organic layer was separated, and the mixture was washed with saturated NaHCO ₃ aqueous solution (2 × 70 mL). The aqueous layers were combined and back-extracted with CHCl ₃ (70 mL). The organic layers were combined, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by chromatography on silica gel to give 12a (829 mg, 90%) as a white foam.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.77 (1H, brs), 7.99 (1H, s), 7.54-6.98 (24H, m), 6.81-6.73 (4H, 4H,) m), 6.35 (1H, dd, J = 8.0, 6.3Hz), 4.89-4.73 (4H, m), 4.68 (2H, brs), 4.05-3. 98 (1H, m), 3.75 (6H, s), 3.62-3.46 (1H, m), 3.41-3.20 (3H, m), 3.18-3.04 ( 1H, m), 3.08 (2H, t, J = 6.6Hz), 2.58-2.36 (2H, m), 1.94-1.59 (2H, m), 1.56 ( 1H, dd, J = 15.0, 8.7Hz), 1.43 (1H, dd, J = 15.0, 5.7Hz), 1.33-1.16 (2H, m), 0.62 (3H, s); ³¹ 1P NMR (121.5 MHz, CDCl ₃ ) δ153.5 (1P, s).

Example Z-11

Oxazaphosphorysine monomer 12b

Compound 12b was obtained in the same manner as compound 12a, but with 3b instead of 3a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.80 (1H, brs), 7.96 (1H, s), 7.54-6.96 (24H, m), 6.79-6.71 (4H, 4H,) m), 6.19 (1H, t, J = 6.6Hz), 4.90-4.73 (4H, m), 4.66 (2H, brass), 4.16-4.08 (1H, m), 3.76 (6H, s), 3.60-3.36 (2H, m), 3.29 (1H, d, J = 3.9Hz), 3.27-3.12 (2H,) m), 3.09 (2H, t, J = 6.6Hz), 2.59-2.46 (1H, m), 2.07-1.97 (1H, m), 1.94-1. 41 (5H, m), 1.36-1.18 (1H, m), 0.65 (3H, s); ³¹ 1P NMR (121.5 MHz, CDCl ₃ ) δ157.1 (1P, s).

Example Z-12

Oxazaphosphorysine monomer 13a

Compound 13a in the same manner as compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr) thymidine. Was obtained using.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.58-7.23 (21H, m), 6.86-6.79 (4H, m), 6.35 (1H, dd, J = 8.1, 5) .7Hz), 4.79-4.67 (2H, m), 3.83-3.78 (1H, m), 3.78 (6H, s), 3.59-3.43 (1H, m) ), 3.34 (1H, dd, J = 10.5, 2.4Hz), 3.35-3.24 (1H, m), 3.20 (1H, dd, J = 10.5, 2. 4Hz), 3.16-3.02 (1H, m), 2.36-2.26 (1H, m), 2.15-2.02 (1H, m), 1.92-1.77 ( 1H, m), 1.74-1.59 (1H, m), 1.52 (1H, dd, J = 14.7, 9.0Hz), 1.40 (3H, s), 1.45- 1.15 (3H, m), 0.60 (3H, s); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ153.7 (1P, s).

Example Z-13

Oxazaphosphoridine monomer 13b

Compound 13b was obtained in the same manner as compound 13a, but with 3b instead of 3a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.46 (1H, brass), 7.59-7.20 (20H, m), 6.86-6.79 (4H, m), 6.26 (1H, 1H, t, J = 6.8Hz), 4.78-4.65 (2H, m), 4.01-3.95 (1H, m), 3.78 (6H, s), 3.55-3. 40 (1H, m), 3.42 (1H, dd, J = 10.5, 2.7Hz), 3.40-3.28 (1H, m), 3.22 (1H, dd, J = 10) .5,3.0Hz), 3.19-3.06 (1H, m), 2.16-1.95 (2H, m), 1.90-1.54 (3H, m), 1.49 -1.35 (1H, m), 1.43 (3H, s), 1.34-1.17 (2H, m), 0.67 (3H, s); ³¹ 1 P NMR (121.5 MHz, CDCl) ₃ ) δ156.2 (1P, s).
Using the above compound 13b, oligos were synthesized by the general methods disclosed above.

Example Z-14

Oxazaphosphorysine monomer 14a

Compound 14a is replaced with 5'O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine in the same manner as compound 12a with 5'O- (DMTr) -4-. Obtained using N- (isobutyryl) cytidine.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.33 (1H, brass), 8.17 (1H, d, J = 7.5 Hz), 7.52-7.22 (19H, m), 7.07 ( 1H, d, J = 7.5Hz), 6.88-6.81 (4H, m), 6.20 (1H, t, J = 6.2Hz), 4.81-4.64 (2H, m) ), 3.93-3.87 (1H, m), 3.79 (6H, s), 3.59-3.43 (1H, m), 3.39-3.29 (3H, m), 3.16-3.02 (1H, m), 2.69-2.52 (2H, m), 2.12-2.00 (1H, m), 1.91-1.50 (3H, m) ), 1.47-1.32 (2H, m), 1.27-1.16 (7H, m), 0.60 (3H, s); ³¹ 1 P NMR (121.5MHz, CDCl ₃ ) δ154. 8 (1P, s).

Example Z-16

Oxazaphosphorysine monomer 14b

Compound 14b was obtained in the same manner as compound 14a, but with 3b instead of 3a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.33 (1H, d, J = 7.5 Hz), 8.23 (1H, brass), 7.57-7.22 (19H, m), 7.12 ( 1H, d, J = 7.5Hz), 6.88-6.81 (4H, m), 6.15 (1H, dd, J = 6.6, 4.2Hz), 4.82-4.63 (2H, m), 4.03-3.97 (1H, m), 3.80 (6H, s), 3.55-3.26 (4H, m), 3.19-3.05 (1H) , M), 2.59 (1H, quantit, J = 6.9Hz), 2.39-2.27 (1H, m), 2.21-2.10 (1H, m), 1.90-1 .56 (3H, m), 1.50-1.32 (2H, m), 1.26-1.17 (7H, m), 0.66 (3H, s); ³¹ 1P NMR (121.5MHz) , CDCl ₃ ) δ157.2 (1P, s).

Example Z-17

Oxazaphosphorysine monomer 15a

Compound 15a is made similar to compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. Obtained using 6-N- (benzoyl) adenosine.

^{1 1} H NMR (600 MHz, CDCl ₃ ) δ8.71 (1H, s), 8.12 (1H, s), 8.04 (2H, d, J = 7.8 Hz), 7.62-7.15 ( 23H, m), 6.80-6.75 (4H, m), 6.37 (1H, dd, J = 7.8, 6.0Hz), 4.94-4.88 (1H, m), 4.80 (1H, ddd, J = 12.0, 6.0, 5.4Hz), 4.07-4.04 (1H, m), 3.76 (6H, s), 3.58-3 .49 (1H, m), 3.41-3.34 (1H, m), 3.33 (1H, dd, J = 10.8, 4.8Hz), 3.25 (1H, dd, J = 10.8, 4.8 Hz), 3.13-3.06 (1H, m), 2.66-2.58 (1H, m), 2.40-2.35 (1H, m), 1. 91-1.84 (1H, m), 1.73-1.66 (1H, m), 1.56 (1H, dd, J = 15.0, 9.0Hz), 1.44 (1H, dd) , J = 15.0, 5.4Hz), 1.47-1.41 (1H, m), 1.30-1.23 (1H, m), 0.63 (3H, s); ³¹ 1 NMR (243.0 MHz, CDCl ³ ) δ151.8 (1P, s).

Example Z-18

Oxazaphosphorysine monomer 15b

Compound 15b was obtained in the same manner as compound 15a, but with 3b instead of 3a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ9.06 (1H, brs), 8.76 (1H, s), 8.12 (1H, s), 8.07-7.99 (2H, m), 7 .64-7.14 (22H, m), 6.83-6.75 (4H, m), 6.25 (1H, t, J = 6.6Hz), 4.86-4.75 (2H,) m), 4.20-4.15 (1H, m), 3.77 (6H, s), 3.61-3.38 (2H, m), 3.36 (1H, dd, J = 10. 2,4.2Hz), 3.27 (1H, dd, J = 10.2, 4.2Hz), 3.27-3.13 (1H, m), 2.71-2.59 (1H, m) ), 2.12-2.01 (1H, m), 1.94-1.42 (5H, m), 1.36-1.20 (1H, m), 0.67 (3H, s)) ³¹ P NMR (121.5 MHz, CDCl ₃ ) δ157.3 (1P, s).

Example Z-19

Oxazaphosphorysine monomer 16a

Compound 16a was obtained in the same manner as compound 13a, but with 7a instead of 3a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.57 (1H, d, J = 0.9 Hz), 7.37-6.94 (20H, m), 6.87-6.78 (4H, m), 6.48 (1H, dd, J = 8.6, 5.7Hz), 5.42 (1H, dd, J = 11.0, 5.1Hz), 4.81-4.71 (1H, m) , 4.02 (1H, d, J = 11.0Hz), 3.83 (1H, d, J = 2.1Hz), 3.79 (6H, s), 3.61-3.41 (2H, m), 3.24-3.09 (1H, m), 3.16 (1H, dd, J = 10.8, 2.4Hz), 3.02 (1H, dd, J = 10.8, 2) .4Hz), 2.54-2.44 (1H, m), 2.34-2.22 (1H, m), 1.94-1.79 (1H, m), 1.74-1.56 (1H, m), 1.38 (3H, s), 1.38-1.28 (2H, m); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ160.9 (1P, s).

Example Z-20

Oxazaphosphorysine monomer 16b

Compound 16b was obtained in the same manner as compound 16a, but with 3b instead of 3a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.57 (1H, d, J = 1.5 Hz), 7.43-7.11 (20H, m), 6.85-6.78 (4H, m), 6.48 (1H, dd, J = 7.5,5.7Hz), 5.58 (1H, dd, J = 11.4,5.1Hz), 4.82-4.73 (1H, m) , 4.17-4.02 (2H, m), 3.78 (6H, s), 3.56-3.40 (3H, m), 3.32 (1H, dd, J = 10.7, 2.4Hz), 3.22-3.07 (1H, m), 2.26-2.04 (2H, m), 1.95-1.81 (1H, m), 1.74-1. 56 (1H, m), 1.40 (3H, d, J = 1.5Hz), 1.44-1.34 (2H, m); ³¹ 1 P NMR (121.5MHz, CDCl ₃ ) δ162.2 ( 1P, s).

Example Z-21

Oxazaphosphorysine monomer 17a

Compound 17a was obtained in the same manner as compound 13a, but with 9a instead of 3a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ9.22 (1H, brass), 8.05-7.99 (2H, m), 7.52 (1H, d, J = 1.2Hz), 7.41- 7.19 (11H, m), 6.87-6.79 (4H, m), 6.37 (1H, dd, J = 8.4,5.7Hz), 4.88-4.75 (2H) , M), 3.86-3.80 (1H, m), 3.79 (6H, d, J = 1.2Hz), 3.64-3.49 (2H, m), 3.27-3 .12 (3H, m), 2.97 (2H, d, J = 6.6Hz), 2.51-2.41 (1H, m), 2.33-2.20 (1H, m), 2 .03-1.75 (2H, m), 1.72-1.59 (1H, m), 1.46-1.36 (1H, m), 1.40 (3H, s); ³¹ 1 NMR (121.5 MHz, CDCl ₃ ) δ157.5 (1P, s).

Example Z-22

Oxazaphosphorysine monomer 17b

Compound 17b was obtained in the same manner as compound 17a, but with 9b instead of 9a.
¹ HN MR (300 MHz, CDCl ₃ ) δ8.67 (1H, brs), 8.18-8.11 (2H, m), 7.57 (1H, d, J = 1.2Hz), 7.47- 7.22 (11H, m), 6.86-6.79 (4H, m), 6.29 (1H, t, J = 6.6Hz), 4.87 (1H, dt, J = 7.5) , 5.7Hz), 4.80-4.72 (1H, m), 4.11-4.05 (1H, m), 3.79 (6H, s), 3.67-3.47 (2H) , M), 3.43 (1H, dd, J = 10.8, 2.7Hz), 3.27 (1H, dd, J = 10.8, 2.4Hz), 3.25-3.13 ( 1H, m), 3.07-2.99 (2H, m), 2.19-2.12 (2H, m), 2.03-1.62 (3H, m), 1.46-1. 30 (1H, m), 1.41 (3H, s); ³¹ P NMR (121.5 MHz, CDCl ₃ ) δ158.1 (1P, s).

Example Z-23

Oxazaphosphorysine monomer 18a

Compound 18a is made similar to compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. Obtained using 2'-O-TOM-6-N- (acetyl) adenosine.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.82 (1H, brs), 8.49 (1H, s), 8.10 (1H, s), 7.58-7.17 (19H, m), 6 .83-6.73 (4H, m), 6.11 (1H, d, J = 6.6Hz), 5.15 (1H, dd, J = 6.6, 5.4Hz), 4.98- 4.77 (4H, m), 4.18-4.11 (1H, m), 3.76 (6H, s), 3.59-3.25 (4H, m), 3.16-3. 02 (1H, m), 2.62 (3H, s), 1.91-1.53 (3H, m), 1.49-1.18 (3H, m), 0.96-0.80 ( 3H, m), 0.90 (18H, s), 0.62 (3H, s); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ156.7 (1P, s).

Example Z-24

Oxazaphosphorysine monomer 18b

Compound 18b was obtained in the same manner as compound 18a, but with 3b instead of 3a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.56 (1H, brs), 8.55 (1H, s), 8.13 (1H, s), 7.57-7.17 (19H, m), 6 .82-6.73 (4H, m), 6.16 (1H, d, J = 5.7Hz), 5.06 (1H, t, J = 5.6Hz), 4.93 (1H, d, J = 5.1Hz), 4.83 (1H, d, J = 5.1Hz), 4.81-4.69 (2H, m), 4.27-4.19 (1H, m), 3. 76 (6H, s), 3.55-3.40 (2H, m), 3.33-3.16 (2H, m), 3.12-2.97 (1H, m), 2.63 ( 3H, s), 1.88-1.52 (3H, m), 1.45-1.16 (3H, m), 0.91-0.79 (3H, m), 0.86 (18H, s), 0.64 (3H, s); ³¹ 1P NMR (121.5 MHz, CDCl ₃ ) δ154.8 (1P, s).

Example Z-25

Oxazaphosphorysine monomer 19a

Compound 19a is made similar to compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. Obtained using 2'-O- (methyl) uridine.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.91 (1H, d, J = 7.8 Hz), 7.58-7.20 (19H, m), 6.88-6.80 (4H, m), 5.96 (1H, d, J = 3.3Hz), 5.19 (1H, d, J = 7.8Hz), 4.88-4.78 (1H, m), 4.66-4.57 (1H, m), 4.03-3.95 (1H, m), 3.90-3.74 (1H, m), 3.78 (6H, s), 3.77-3.71 (1H) , M), 3.58-3.29 (2H, m), 3.45 (3H, s), 3.13-2.82 (2H, m), 1.88-1.53 (3H, m) ), 1.49-1.16 (3H, m), 0.60 (3H, s); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ155.3 (1 P, s).

Example Z-26

Oxazaphosphorysine monomer 19b

Compound 19b was obtained in the same manner as compound 19a, but with 3b instead of 3a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.10 (1H, d, J = 8.4 Hz), 7.58-7.20 (19H, m), 6.87-6.79 (4H, m), 5.89 (1H, d, J = 1.5Hz), 5.21 (1H, d, J = 8.4Hz), 4.92-4.82 (1H, m), 4.73-4.63 (1H, m), 4.15-4.08 (1H, m), 3.89-3.73 (1H, m), 3.78 (6H, s), 3.66-3.62 (1H) , M), 3.57-3.27 (2H, m), 3.30 (3H, s), 3.17-2.82 (2H, m), 1.89-1.55 (3H, m) ), 1.55-1.40 (1H, m), 1.35-1.15 (2H, m), 0.66 (3H, s); ³¹ 1 P NMR (121.5MHz, CDCl ₃ ) δ157. 5 (1P, s).

Example Z-27

Oxazaphosphorysine monomer 20a

Compound 20a is made similar to compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. Obtained using 2'-deoxy-2'-fluorouridine.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.85 (1H, d, J = 8.1 Hz), 7.58-7.20 (19H, m), 6.87-6.79 (4H, m), 5.98 (1H, d, J = 16.5Hz), 5.23 (1H, d, J = 8.1Hz), 4.86-4.61 (3H, m), 3.99 (1H, d) , J = 6.9Hz), 3.76 (6H, d, J = 3.0Hz), 3.56-3.34 (4H, m), 3.10-2.96 (1H, m), 1 .88-1.74 (1H, m), 1.72-1.52 (2H, m), 1.48-1.16 (3H, m), 0.61 (3H, s); ³¹ 1 NMR (121.5 MHz, CDCl ₃ ) δ154.3 (1P, d, J = 8.9 Hz).

Example Z-28

Oxazaphosphorysine monomer 20b

Compound 20b was obtained in the same manner as compound 20a, but with 3b instead of 3a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.01 (1H, d, J = 8.4 Hz), 7.58-7.20 (19H, m), 6.87-6.79 (4H, m), 6.03 (1H, d, J = 16.2Hz), 5.29 (1H, d, J = 8.4Hz), 4.96 (1H, dd, J = 13.1,7.5Hz), 4 .80-4.54 (2H, m), 4.15 (1H, d, J = 9.0Hz), 3.78 (6H, s), 3.61-3.39 (3H, m), 3 .37-3.25 (1H, m), 3.23-3.09 (1H, m), 1.91-1.56 (3H, m), 1.51-1.13 (3H, m) , 0.66 (3H, s); ³¹ 1P NMR (121.5 MHz, CDCl ₃ ) δ158.9 (1P, d, J = 4.4 Hz).

Example Z-29

Oxazaphosphorysine monomer 21a

Compound 21a is made similar to compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. Obtained using 2'-O-methoxyethyl-5-methyluridine.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.62-7.18 (21H, m), 6.84 (4H, d, J = 8.7Hz), 6.07 (1H, d, J = 5.7Hz) ), 4.86-4.76 (1H, m), 4.63-4.54 (1H, m), 4.20 (1H, t, J = 5.4Hz), 3.95-3.89 (1H, m), 3.78 (6H, s), 3.78-3.71 (2H, m), 3.60-3.48 (2H, m), 3.44-3.02 (5H) , M), 3.31 (3H, s), 1.88-1.15 (6H, m), 1.35 (3H, s), 0.58 (3H, s); ³¹ 1P NMR (121. 5 MHz, CDCl ₃ ) δ156.3 (1P, s).

Example Z-30

Oxazaphosphorysine monomer 21b

Compound 21b was obtained in the same manner as compound 21a, but with 3b instead of 3a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.71 (1H, d, J = 1.2 Hz), 7.55-7.22 (20H, m), 6.86-6.78 (4H, m), 5.99 (1H, d, J = 3.9Hz), 4.78-4.62 (2H, m), 4.13-4.08 (1H, m), 4.07-4.02 (1H) , M), 3.77 (6H, s), 3.77-3.70 (1H, m), 3.65-3.56 (1H, m), 3.52-3.36 (4H, m) ), 3.33-3.14 (2H, m), 3.29 (3H, s), 3.08-2.94 (1H, m), 1.86-1.72 (1H, m), 1.71-1.55 (2H, m), 1.30 (3H, d, J = 1.2Hz), 1.47-1.16 (3H, m) 0.64 (3H, s); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ155.6 (1P, s).

Example Z-31

Oxazaphosphorysine monomer 22a

Compound 22a is made similar to compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. Obtained using 2'-O-methyl-4-N- (isobutyryl) cytidine.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ 8.49 (1 H, d, J = 7.2 Hz), 7.58-7.20 (19 H, m), 6.96 (1 H, d, J = 7.2 Hz) ), 6.90-6.82 (4H, m), 5.98 (1H, s), 4.84 (1H, dd, J = 13.1,7.5Hz), 4.59 (1H, dt) , J = 8.3,4.5Hz), 4.19-4.13 (1H, m), 3.79 (6H, s), 3.78-3.72 (1H, m), 3.63 -3.40 (3H, m), 3.55 (3H, s), 3.36-3.24 (1H, m), 3.09-2.95 (1H, m), 2.59 (1H) , Seven-fold wire, J = 6.9Hz), 1.85-1.53 (5H, m), 1.48-1.37 (1H, m), 1.24-1.17 (6H, m), 0.59 (3H, s); ³¹ 1P NMR (121.5 MHz, CDCl ₃ ) δ155.2 (1P, s).

Example Z-32

Oxazaphosphorysine monomer 22b

Compound 22b was obtained in the same manner as compound 22a, but with 3b instead of 3a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.62 (1H, d, J = 7.5 Hz), 7.57-7.23 (19H, m), 7.02 (1H, d, J = 7.5 Hz) ), 6.89-6.81 (4H, m), 5.92 (1H, s), 4.90 (1H, dt, J = 9.0, 5.7Hz), 4.61 (1H, dt) , J = 8.7, 4.8Hz), 4.25-4.17 (1H, m), 3.81 (6H, s), 3.67 (1H, d, J = 4.5Hz), 3 .62-3.25 (4H, m), 3.38 (3H, s), 3.16-3.02 (1H, m), 2.58 (1H, septet, J = 6.9Hz), 1 .87-1.40 (6H, m), 1.26-1.14 (6H, m), 0.64 (3H, s); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ158.2 (1P) , S).

Example Z-33

Oxazaphosphorysine monomer 23a

Compound 23a is made similar to compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. Obtained using 2'-O-methyl-2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.67 (1H, brs), 8.01 (1H, s), 7.56-7.16 (24H, m), 6.83-6.74 (4H, 4H,) m), 6.08 (1H, d, J = 6.9Hz), 4.85-4.76 (1H, m), 4.84 (2H, t, J = 6.6Hz), 4.65- 4.56 (1H, m), 4.59 (2H, brass), 4.48 (1H, dd, J = 6.6, 5.1Hz), 4.09-4.05 (1H, m), 3.75 (6H, s), 3.60-3.42 (2H, m), 3.40-3.26 (2H, m), 3.35 (3H, s), 3.18-3. 05 (1H, m), 3.08 (2H, t, J = 6.6Hz), 1.89-1.49 (3H, m), 1.48-1.16 (3H, m), 0. 59 (3H, s); ³¹ 1P NMR (121.5 MHz, CDCl ₃ ) δ156.9 (1P, s).

Example Z-34

Oxazaphosphorysine monomer 23b

Compound 23b was obtained in the same manner as compound 23a, but with 3b instead of 3a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.74 (1H, brs), 8.09 (1H, s), 7.56-6.94 (24H, m), 6.84-6.71 (4H, s). m), 6.09 (1H, d, J = 4.8Hz), 4.83-4.70 (2H, m), 4.83 (2H, t, J = 6.6Hz), 4.63 ( 2H, brass), 4.35 (1H, t, J = 5.0Hz), 4.23-4.16 (1H, m), 3.75 (6H, s), 3.58-3.19 ( 4H, m), 3.32 (3H, s), 3.16-3.04 (1H, m), 3.07 (2H, t, J = 6.6Hz), 1.90-1.55 ( 3H, m), 1.48-1.15 (3H, m), 0.64 (3H, s); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ154.6 (1 P, s).

Example Z-35

Oxazaphosphorysine monomer 24a

Compound 24a is made similar to compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. It was obtained using 2'-deoxy-2'-fluoro-2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.74 (1H, brass), 8.03 (1H, s), 7.55-6.94 (24H, m), 6.80-6.69 (4H, 4H,) m), 6.21 (1H, dd, J = 14.9, 3.6Hz), 5.34 (1H, dt, J = 52.3, 3.6Hz), 5.01-4.75 (2H) , M), 4.84 (1H, t, J = 6.6Hz), 4.62 (2H, brass), 4.15-4.07 (1H, m), 3.73 (6H, s), 3.59-3.29 (4H, m), 3.15-3.00 (1H, m), 3.07 (2H, t, J = 6.6Hz), 1.90-1.49 (3H) , M), 1.47-1.12 (3H, m), 0.58 (3H, s); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ155.6 (1P, d, J = 10.9 Hz) ).

Example Z-36

Oxazaphosphorysine monomer 24b

Compound 24b was obtained in the same manner as compound 24a, but with 3b instead of 3a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.81 (1H, brass), 8.06 (1H, s), 7.55-6.95 (24H, m), 6.77-6.69 (4H, 4H,) m), 6.06 (1H, d, J = 17.1Hz), 5.24-5.08 (1H, m), 5.04-4.80 (2H, m), 4.87 (1H, t, J = 6.6Hz), 4.62 (2H, brs), 4.25-4.19 (1H, m), 3.73 (6H, s), 3.58-3.02 (5H, m), 3.10 (2H, t, J = 6.6Hz), 1.90-1.56 (3H, m), 1.50-1.15 (3H, m), 0.63 (3H, s); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ158.0 (1P, d, J = 4.4 Hz).

Example Z-37

Oxazaphosphorysine monomer 25a

Compound 25a is made similar to compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. Obtained using 2'-O-TOM-4-N- (acetyl) cytidine.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ10.04 (1H, brass), 8.30 (1H, d, J = 7.5 Hz), 7.51-7.21 (19H, m), 6.99 ( 1H, d, J = 7.5Hz), 6.89-6.81 (4H, m), 6.12 (1H, d, J = 3.3Hz), 5.07 (1H, d, J = 4) .8Hz), 5.05 (1H, d, J = 4.8Hz), 4.84-4.75 (1H, m), 4.62-4.52 (1H, m), 4.31-4 .25 (1H, m), 4.08-4.01 (1H, m), 3.78 (6H, d, J = 3.0Hz), 3.55-3.23 (4H, m), 3 .10-2.96 (1H, m), 2.24 (3H, s), 1.84-1.49 (3H, m), 1.46-0.96 (24H, m), 0.58 (3H, s); ³¹ 1P NMR (121.5 MHz, CDCl ₃ ) δ156.5 (1P, s).

Example Z-38

Oxazaphosphorysine monomer 25b

Compound 25b was obtained in the same manner as compound 25a, but with 3b instead of 3a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ10.19 (1H, brass), 8.46 (1H, d, J = 7.5 Hz), 7.54-7.23 (19H, m), 7.01 ( 1H, d, J = 7.5Hz), 6.88-6.79 (4H, m), 6.19 (1H, d, J = 1.8Hz), 5.11 (1H, d, J = 4) .8Hz), 5.07 (1H, d, J = 4.8Hz), 4.81-4.71 (1H, m), 4.60-4.51 (1H, m), 4.26-4 .18 (2H, m), 3.79 (6H, s), 3.63-3.55 (1H, m), 3.48-3.28 (2H, m), 3.21-2.94 (2H, m), 2.26 (3H, s), 1.81-1.49 (3H, m), 1.43-0.96 (24H, m), 0.62 (3H, s); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ156.4 (1P, s).

Example Z-39

Oxazaphosphorysine monomer 26a

Compound 26a is made similar to compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. Obtained using 2'-deoxy-2'-fluoro-4-N- (isobutyryl) cytidine.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.66 (1H, brass), 8.41 (1H, d, J = 7.5 Hz), 7.55-7.20 (19H, m), 7.01 ( 1H, d, J = 7.5Hz), 6.89-6.81 (4H, m), 6.06 (1H, d, J = 15.9Hz), 4.85 (1H, dd, J = 51) .4,3.9Hz), 4.84 (1H, dd, J = 12.9, 7.5Hz), 4.77-4.59 (1H, m), 4.15-4.08 (1H, 1H, m), 3.79 (6H, s), 3.63-3.29 (4H, m), 3.10-2.96 (1H, m), 2.65 (1H, septet, J = 6. 9Hz), 1.85-1.53 (3H, m), 1.48-1.17 (3H, m), 1.21 (3H, d, J = 4.8Hz), 1.19 (3H, d, J = 4.8 Hz), 0.59 (3H, s); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ155.5 (1 P, d, J = 6.6 Hz).

Example Z-40

Oxazaphosphorysine monomer 26b

Compound 26b was obtained in the same manner as compound 26a, but with 3b instead of 3a.
^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.53 (1H, d, J = 7.5 Hz), 7.57-7.23 (20H, m), 7.10 (1H, d, J = 7.5 Hz) ), 6.89-6.81 (4H, m), 6.10 (1H, d, J = 15.9Hz), 5.00-4.92 (1H, m), 4.84 (1H, dd) , J = 51.5, 3.3Hz), 4.75-4.58 (1H, m), 4.24 (1H, d, J = 9.3Hz), 3.81 (6H, s), 3 .65-3.39 (3H, m), 3.32-3.06 (2H, m), 2.59 (1H, septet, J = 6.9Hz), 1.88-1.53 (4H, m), 1.49-1.34 (2H, m), 1.27-1.18 (6H, m), 0.65 (3H, s); ³¹ 1 P NMR (121.5MHz, CDCl ₃ ) δ159 .0 (1P, d, J = 4.4).

Oxazaphosphorysine monomer 27a

Compound 27a is made similar to compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. Obtained using 2'-O-methyl-6-N- (benzoyl) adenosine.

^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.66 (1H, s), 8.13 (1H, s), 8.03 (2H, d, J = 7.2 Hz), 7.64-7.16 ( 23H, m), 6.79 (4H, d, J = 8.7Hz), 6.08 (1H, d, J = 6.3Hz), 4.91-4.81 (1H, m), 4. 77-4.69 (1H, m), 4.64-4.57 (1H, m), 4.15-4.10 (1H, m), 3.76 (6H, s), 3.60- 3.23 (4H, m), 3.35 (3H, s), 3.14-3.00 (1H, m), 1.90-1.19 (6H, m), 0.62 (3H, 3H,) s); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ155.8 (1P, s).

Oxazaphosphorysine monomer 27b

Compound 27b was obtained in the same manner as compound 27a, but with 3b instead of 3a.

^{1 1} H NMR (300 MHz, CDCl ₃ ) δ9.12 (1H, brs), 8.73 (1H, s), 8.24 (1H, s), 8.07-8.01 (2H, m), 7 .62-7.17 (22H, m), 6.83-6.77 (4H, m), 6.12 (1H, d, J = 4.8Hz), 4.84-4.73 (2H, m), 4.43 (1H, t, J = 4.8Hz), 4.25-4.19 (1H, m), 3.77 (6H, s), 3.55-3.20 (4H, m), 3.28 (3H, s), 3.16-3.03 (1H, m), 1.90-1.17 (6H, m), 0.65 (3H, s); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ155.0 (1P, s).

Oxazaphosphorysine monomer 28a

Compound 28a, like compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. Obtained using 2'-deoxy-2'-fluoro-6-N- (benzoyl) adenosine.

^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.64 (1H, s), 8.14 (1H, s), 8.06-8.01 (2H, m), 7.63-7.07 (23H, 23H,) m), 6.78-6.70 (4H, m), 6.12 (1H, dd, J = 18.0, 2.4Hz), 5.24-5.01 (2H, m), 4. 94-4.84 (1H, m), 4.17-4.06 (1H, m), 3.73 (6H, s), 3.55-3.40 (3H, m), 3.30- 3.22 (1H, m), 3.03-2.88 (1H, m), 1.92-1.19 (6H, m), 0.62 (3H, s); ³¹ 1 P NMR (121. 5 MHz, CDCl ₃ ) δ150.5 (1P, d, J = 7.7 Hz).

Oxazaphosphorysine monomer 28b

Compound 28b was obtained in the same manner as compound 28a, but with 3b instead of 3a.

^{1 1} H NMR (300 MHz, CDCl ₃ ) δ9.07 (1H, brs), 8.80 (1H, s), 8.24 (1H, s), 8.08-8.01 (2H, m), 7 .66-7.15 (22H, m), 6.81-6.75 (4H, m), 6.14 (1H, dd, J = 18.0, 1.8Hz), 5.16-4. 91 (3H, m), 4.28-4.21 (1H, m), 3.76 (6H, s), 3.57-3.11 (5H, m), 1.82-1.16 ( 6H, m), 0.65 (3H, s); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ157.8 (1P, d, J = 5.6 Hz).

Oxazaphosphorysine monomer 29a

Compound 29a is made similar to compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. Obtained using 2'-O-TOM-2-N- (acetyl) guanosine.

^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.70 (1H, s), 7.63-7.13 (21H, m), 6.84-6.76 (4H, m), 5.77 (1H, 1H, d, J = 8.4 Hz), 5.41-5.33 (1H, m), 4.90 (2H, s), 4.78-4.68 (2H, m), 3.86 (1H, 1H,) Brs), 3.75 (3H, s), 3.74 (3H, s), 3.56-3.41 (2H, m), 3.32-2.90 (3H, m), 1.92 -1.10 (9H, m), 0.97-0.87 (21H, m), 0.52 (3H, s); ³¹ 1P NMR (121.5MHz, CDCl ₃ ) δ158.1 (1P, s) ).

Oxazaphosphorysine monomer 29b

Compound 29b was obtained in the same manner as compound 29a, but with 3b instead of 3a.

^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.77 (1H, s), 7.56-7.15 (21H, m), 6.82-6.75 (4H, m), 5.86 (1H, 1H,) d, J = 7.5Hz), 5.26-5.17 (1H, m), 4.95 (1H, d, J = 5.4Hz), 4.85 (1H, d, J = 5.4Hz) ), 4.78-4.71 (1H, m), 4.59-4.49 (1H, m), 4.10-4.05 (1H, m), 3.74 (6H, s), 3.52-3.37 (2H, m), 3.30-1.18 (1H, m), 3.11-2.85 (2H, m), 1.85-1.15 (9H, m) ), 0.93-0.84 (21H, m), 0.62 (3H, s); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ152.3 (1 P, s).

Oxazaphosphorysine monomer 30a

Compound 30a is made similar to compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. Obtained using 2'-O-TOM-uridine.

^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.76 (1H, d, J = 8.1 Hz), 7.55-7.18 (20H, m), 6.88-6.80 (4H, m), 6.11 (1H, d, J = 6.0Hz), 5.32 (1H, d, J = 8.1Hz), 4.99 (1H, d, J = 5.1Hz), 4.93 (1H) , D, J = 5.1Hz), 4.84-4.75 (1H, m), 4.54-4.46 (1H, m), 4.38 (1H, t, J = 5.7Hz) , 3.87-3.83 (1H, m), 3.78 (3H, s), 3.77 (3H, s), 3.56-3.42 (1H, m), 3.39-3 .28 (1H, m), 3.36 (1H, dd, J = 11.0, 2.7Hz), 3.25 (1H, dd, J = 11.0, 2.7Hz), 3.16- 3.03 (1H, m), 1.88-1.12 (6H, m), 1.08-0.97 (21H, m), 0.59 (3H, s); ³¹ 1P NMR (121. 5 MHz, CDCl ₃ ) δ156.6 (1P, s).

Oxazaphosphorysine monomer 30b

Compound 30b was obtained in the same manner as compound 30a, but with 3b instead of 3a.

^{1 1} H NMR (600 MHz, CDCl ₃ ) δ7.87 (1H, d, J = 7.8 Hz), 7.52-7.48 (4H, m), 7.38-7.21 (16H, m), 6.83-6.79 (4H, m), 6.14 (1H, d, J = 4.8Hz), 5.33 (1H, d, J = 7.8Hz), 4.99 (1H, d) , J = 5.4Hz), 4.89 (1H, d, J = 5.4Hz), 4.67 (1H, dd, J = 13.8, 7.2Hz), 4.52 (1H, dt, J = 10.4, 4.8Hz), 4.31 (1H, t, J = 4.8Hz), 4.06-4.03 (1H, m), 3.78 (3H, s), 3. 77 (3H, s), 3.47 (1H, dd, J = 10.4, 2.4Hz), 3.47-3.39 (1H, m), 3.22-3.17 (2H, m) ), 3.00 (1H, ddd, J = 19.5, 10.4, 4.8Hz), 1.82-1.74 (1H, m), 1.68-1.58 (1H, m) , 1.56 (1H, dd, J = 14.4, 8.4Hz), 1.38 (1H, dd, J = 14.4, 7.2Hz), 1.31-1.25 (1H, m) ), 1.26-1.17 (1H, m), 1.08-0.98 (21H, m), 0.63 (3H, s); ³¹ 1 P NMR (243.0 MHz, CDCl ₃ ) δ154. 3 (1P, s).

Oxazaphosphorysine monomer 31a

Compound 31a is made similar to compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. Obtained using 2'-O, 4'-C-methylene-6-N- (benzoyl) adenosine.

^{1 1} H NMR (300 MHz, CDCl ₃ ) δ9.10 (1H, brs), 8.76 (1H, s), 8.32 (1H, s), 8.04 (2H, d, J = 7.2 Hz) , 7.64-7.18 (22H, m), 6.84 (4H, d, J = 8.7Hz), 6.10 (1H, s), 4.76 (1H, dJ = 6.9Hz) , 4.58 (1H, s), 4.61-4.51 (1H, m), 3.91 (1H, d, J = 7.8Hz), 3.77 (1H, d, J = 7. 8Hz), 3.75 (6H, s), 3.50 (1H, s), 3.47-3.33 (1H, m), 3.31-3.19 (1H, m), 3.03 -2.88 (1H, m), 1.84-1.09 (6H, m), 0.51 (3H, s); ³¹ 1P NMR (121.5MHz, CDCl ₃ ) δ152.9 (1P, s) ).

Oxazaphosphorysine monomer 31b

Compound 31b was obtained in the same manner as compound 31a, but with 3b instead of 3a.

^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.81 (1H, s), 8.30 (1H, s), 8.07-8.00 (2H, m), 7.64-7.17 (22H, 22H,) m), 6.86-6.79 (4H, m), 6.12 (1H, s), 4.81-4.72 (1H, m), 4.62 (1H, dJ = 7.2Hz) , 4.57 (1H, s), 3.94 (1H, d, J = 7.8Hz), 3.89 (1H, d, J = 7.8Hz), 3.77 (6H, s), 3 .48 (2H, s), 3.46-3.32 (1H, m), 3.24-3.13 (1H, m), 3.10-2.97 (1H, m), 1.84 -1.49 (3H, m), 1.42-1.09 (3H, m), 0.58 (3H, s); ³¹ 1P NMR (121.5MHz, CDCl ₃ ) δ157.3 (1P, s) ).

Oxazaphosphorysine monomer 32a

Compound 32a, like compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. It was obtained using 2'-O, 4'-C-methylene-4-N- (isobutyryl) -5-methylcytidine.

^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.88 (1H, brass), 7.58-7.18 (20H, m), 6.88-6.80 (4H, m), 5.65 (1H, 1H, s), 4.69-4.60 (1H, m), 4.52 (1H, d, J = 6.6Hz), 4.49 (1H, s), 3.81-3.74 (1H, 1H, m), 3.75 (3H, s), 3.73 (3H, s), 3.64 (1H, d, J = 8.1Hz), 3.56 (1H, d, J = 11.1Hz) , 3.53 (1H, d, J = 8.1Hz), 3.46 (1H, d, J = 11.1Hz), 3.56-3.40 (1H, m), 3.32-3. 20 (1H, m), 3.14-3.00 (1H, m), 1.85-1.12 (6H, m), 1.60 (3H, s), 1.19 (6H, d, J = 6.9 Hz), 0.55 (3H, s); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ155.9 (1 P, s).

Oxazaphosphorysine monomer 32b

Compound 32b was obtained in the same manner as compound 32a, but with 3b instead of 3a.

^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.86 (1H, brass), 7.56-7.19 (20H, m), 6.88-6.79 (4H, m), 5.69 (1H, 1H, s), 4.86-4.76 (1H, m), 4.46 (1H, s), 4.45 (1H, d, J = 7.5Hz), 3.80-3.75 (1H, m), 3.79 (6H, s), 3.74 (1H, d, J = 8.1Hz), 3.69 (1H, d, J = 8.1Hz), 3.51 (1H, d, J = 11.1Hz), 3.44-3.30 (1H, m), 3.39 (1H, d, J = 11.1Hz), 3.29-3.17 (1H, m), 3. 11-2.97 (1H, m), 1.86-1.52 (3H, m), 1.64 (3H, s), 1.45-1.10 (3H, m), 1.21 ( 6H, d, J = 6.6Hz), 0.62 (3H, s); ³¹ 1P NMR (121.5MHz, CDCl ₃ ) δ158.2 (1P, s).

Oxazaphosphorysine monomer 33a

Compound 33a is made similar to compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. It was obtained using 2'-O, 4'-C-methylene-2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine.

^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.71 (1H, brs), 8.16 (1H, s), 7.50-7.17 (21H, m), 7.09-7.01 (3H, 3H,) m), 6.86-6.79 (4H, m), 6.03 (1H, s), 4.84 (2H, t, J = 6.6Hz), 4.72 (2H, s), 4 .68 (1H, d, J = 7.2Hz), 4.55-4.46 (1H, m), 4.50 (1H, s), 3.90 (1H, d, J = 7.8Hz) , 3.77 (1H, d, J = 7.8Hz), 3.75 (6H, s), 3.51 (1H, d, J = 10.8Hz), 3.47 (1H, d, J =) 10.8Hz), 3.45-3.21 (2H, m), 3.08 (2H, t, J = 6.6Hz), 3.03-2.89 (1H, m), 1.80- 1.08 (6H, m), 0.47 (3H, s); ³¹ 1P NMR (121.5 MHz, CDCl ₃ ) δ153.2 (1P, s).

Oxazaphosphorysine monomer 33b

Compound 33b was obtained in the same manner as compound 33a, but with 3b instead of 3a.

^{1 1} H NMR (300 MHz, CDCl ₃ ) δ8.86 (1H, brs), 8.13 (1H, s), 7.55-7.17 (21H, m), 7.08-6.98 (3H, 3H,) m), 6.95-6.78 (4H, m), 6.01 (1H, s), 4.86 (2H, t, J = 6.6Hz), 4.82-4.73 (1H, m), 4.70 (2H, s), 4.64 (1H, d, J = 7.5Hz), 4.49 (1H, s), 3.94 (1H, d, J = 7.8Hz) , 3.89 (1H, d, J = 7.8Hz), 3.77 (6H, s), 3.46 (2H, s), 3.45-3.30 (1H, m), 3.24 -3.12 (1H, m), 3.09 (2H, t, J = 6.6Hz), 3.09-2.96 (1H, m), 1.81-1.50 (3H, m) , 1.41-1.06 (3H, m), 0.58 (3H, s); ³¹ 1P NMR (121.5 MHz, CDCl ₃ ) δ157.4 (1P, s).

Oxazaphosphorysine monomer 34a

Compound 34a is made similar to compound 12a, but instead of 5'-O- (DMTr) -2-N- (phenoxyacetyl) -6-O- (cyanoethyl) guanosine, 5'-O- (DMTr)-. Obtained using 2'-O, 4'-C-methylene-5-methyluridine.

^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.71 (1H, d, J = 0.9 Hz), 7.50-7.17 (20H, m), 6.87-6.80 (4H, m), 5.61 (1H, s), 4.69-4.60 (1H, m), 4.55 (1H, d, J = 6.9Hz), 4.41 (1H, s), 3.74 ( 3H, s), 3.73 (3H, s), 3.64 (1H, d, J = 7.8Hz), 3.55 (1H, d, J = 7.8Hz), 3.53 (1H, d, J = 10.8Hz), 3.46 (1H, d, J = 10.8Hz), 3.56-3.42 (1H, m), 3.35-3.24 (1H, m), 3.13-3.00 (1H, m), 1.85-1.45 (3H, m), 1.55 (3H, d, J = 0.9Hz), 1.41-1.12 (3H) , M), 0.56 (3H, s); ³¹ 1P NMR (121.5 MHz, CDCl ₃ ) δ155.1 (1P, s).

Oxazaphosphorysine monomer 34b

Compound 34b was obtained in the same manner as compound 34a, but with 3b instead of 3a.

^{1 1} H NMR (300 MHz, CDCl ₃ ) δ7.69 (1H, s), 7.56-7.19 (20H, m), 6.88-6.79 (4H, m), 5.66 (1H, 1H,) s), 4.87-4.77 (1H, m), 4.47 (1H, d, J = 7.8Hz), 4.40 (1H, s), 3.78 (6H, s), 3 .74 (1H, d, J = 7.8Hz), 3.68 (1H, d, J = 7.8Hz), 3.50 (1H, d, J = 10.8Hz), 3.46-3. 32 (1H, m), 3.39 (1H, d, J = 10.8Hz), 3.30-3.19 (1H, m), 3.12-2.98 (1H, m), 1. 85-1.56 (3H, m), 1.59 (3H, s), 1.46-1.12 (3H, m), 0.63 (3H, s); ³¹ 1 P NMR (121.5 MHz, CDCl ₃ ) δ158.1 (1P, s).

Oxazaphosphorysine monomer 35a

Compound 35a was obtained in the same manner as compound 13a, but with 13a'instead of 3a. ^{1 1} H NMR (600 MHz, CDCl ₃ ) δ7.76 (2H, d, J = 9.0 Hz), 7.62 (1H, d, J = 1.2 Hz), 7.40 (2H, d, J = 7) .2Hz), 6.32-7.23 (10H, m), 6.85 (4H, d, J = 8.4Hz), 6.41 (1H, dd, J = 8.4,5.4Hz) , 4.94 (1H, dd, J = 12.3, 5.4Hz), 4.84-4.79 (1H, m), 4.03-4.01 (1H, m), 3.79 ( 6H, s), 3.59-3.53 (1H, m), 3.52-3.44 (2H, m), 3.41 (1H, dd, J = 14.7, 7.2Hz), 3.37-3.30 (2H, m), 3.13 (1H, ddd, J = 19.3, 10.3, 4.1Hz), 2.50-2.44 (1H, m), 2 .39 (3H, s), 2.35-2.29 (1H, m), 1.91-1.72 (2H, m), 1.64-1.59 (1H, m), 1.40 (3H, s), 1.12-1.05 (1H, m); ³¹ 1 P NMR (243.0 MHz, CDCl ₃ ) δ154.2 (1 P, s).

Example Z-41

The above compound Z-27 represents a conventional monomer, which is used to produce an oligo. FIG. 70 shows a chart of products obtained through Comparative Example Z-1. As shown in FIGS. 69 and 70, the monomers of the invention provided more complete deprotection and less by-products, which facilitates product isolation and / or purification.

In some embodiments, the invention provides a chemically stable monomer. Exemplary such monomers are shown above in the examples. In some embodiments, the invention provides monomers with high isolation yields. In some embodiments, the invention provides monomers with higher isolation yields than conventional methods. In some embodiments, the isolated yield is greater than 80%. Exemplary such monomers are shown above in the examples.

、
式中、Ｚ^１、Ｚ^２、Ｚ^３、Ｚ^４、Ｚ^５、Ｚ^６、Ｚ^７、Ｚ^８、及びＺ^９は、互いに独立してアルキル、アミノアルキル、シクロアルキル、複素環、シクロアルキルアルキル、ヘテロシクロアルキル、アリール、ヘテロアリール、アルキルオキシ、アリールオキシ、若しくはヘテロアリールオキシより選択される、任意に置換された置換基であってもよく、あるいは、Ｚ^２とＺ^３、Ｚ^５とＺ^６、Ｚ^７とＺ^８、Ｚ^８とＺ^９、Ｚ^９とＺ^７、若しくはＺ^７とＺ^８とＺ^９、のいずれかの組み合わせにより３～２０員環脂環式環又は複素環を形成し；Ｑ^－は対アニオンであり、ＬＧは脱離基である。 Condensation Reagent A condensation reagent ( _CR ) useful according to the method of the invention may be any of the following general formulas:

,
In the formula, Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ , Z ⁶ , Z ⁷ , Z ⁸ and Z ⁹ are alkyl, aminoalkyl, cycloalkyl, heterocycle, cycloalkylalkyl independently of each other. , Heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, or optionally substituted substituents selected from heteroaryloxy, or Z ² and Z ³ , Z ⁵ and Z. A 3- to 20-membered alicyclic ring or heterocycle is formed by any combination of ⁶ , Z ⁷ and Z ⁸ , Z ⁸ and Z ⁹ , Z ⁹ and Z ⁷ , or Z ⁷ and Z ⁸ and Z ⁹ . Q ^- is a counter-anion and LG is a leaving group.

In some embodiments, the counterion of the condensation reagent _CR is Cl ⁻ , Br ⁻ , BF ₄ ⁻ , PF ₆ ⁻ , TfO ⁻ , Tf ₂ N ⁻ , AsF ₆ ⁻ , ClO ₄ ⁻ , or SbF ₆ ⁻ . Yes, Tf is CF ₃ SO ₂ . In some embodiments, the leaving group of the condensation reagent _CR is F, Cl, Br, I, 3-nitro-1,2,4-triazole, imidazole, alkyltriazole, tetrazole, pentafluorobenzene, or 1-. Hydroxybenzotriazole.

Examples of condensing reagents used according to the method of the present invention include pentafluorobenzoyl chloride, carbonyldiimidazole (CDI), 1-methicylene sulphonyl-3-nitrotriazole (MSNT), 1-ethyl-3- (3'-dimethylamino). Propyl) Carbodiimide Hydrochloride (EDCI-HCl), Bentriazole-1-yloxytris (Dimethylamino) Phosphorium Hexafluorophosphate (PyBOP), N, N'-bis (2-oxo-3-oxazolidinyl) Phosphinic acid chloride (2-oxo-3-oxazolidinyl) phosphinic acid chloride (PyBOP) BopCl), 2- (1H-7-azabenzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), and O-benzotriazole-N, N, N, 'N'-Tetramethyluronium Hexafluorophosphate (HBTU), DIPCDI; N, N'-bis (2-oxo-3-oxazolidinyl) phosphinic acid bromide (BopBr), 1,3-dimethyl-2- (3-) Nitro-1,2,4-triazole-1-yl) -2-pyrrolidin-1-yl-1,3,2-diazaphosphoridinium hexafluorophosphate (MNTP), 3-nitro-1,2,4 -Triazole-1-yl-tris (pyrrolidin-1-yl) phosphonium hexafluorophosphate (PyNTP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP); O- (benzotriazole-1-yl) -N, N, Examples include, but are not limited to, N', N'-tetramethyluronium tetrafluoroborate (TBTU); and tetramethylfluoroformamidinium hexafluorophosphate (TFFH). In certain embodiments, the counterion of the condensation reagent _CR is Cl ⁻ , Br ⁻ , BF ₆ ⁻ , PF ₆ ⁻ , TfO ⁻ , Tf ₂ N ⁻ , AsF ₆ ⁻ , ClO ₄ ⁻ , or SbF ₆ ⁻ . Yes, Tf is CF ₃ SO ₂ .

In some embodiments, the condensing reagent is 1- (2,4,6-triisopropylbenzenesulfonyl) -5- (pyridin-2-yl) tetrazolide, pivaloyl chloride, bromotrispyrrolidinophosphonium hexafluorophosphate. , N, N'-bis (2-oxo-3-oxazolidinyl) phosphinic acid chloride (BopCl), or 2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinan. .. In some embodiments, the condensing reagent is an N, N'-bis (2-oxo-3-oxazolidinyl) phosphinic acid chloride (BopCl). In some embodiments, the condensing reagent is selected from those described in WO2006 / 066260.

である。 In some embodiments, the condensing reagent is 1,3-dimethyl-2- (3-nitro-1,2,4-triazole-1-yl) -2-pyrrolidin-1-yl-1,3,2-. Diazaphosphoridinium hexafluorophosphate (MNTP) or 3-nitro-1,2,4-triazole-1-yl-tris (pyrrolidin-1-yl) phosphonium hexafluorophosphate (PyNTP):

Is.

Selection of Bases and Sugars for Nucleoside Coupling Partners As described herein, the nucleoside coupling partners used according to the methods of the invention may be the same or different from each other. In some embodiments, the nucleotide coupling partners used in the synthesis of the provided oligonucleotides have the same structure and / or stereochemical arrangement with each other. In some embodiments, each nucleoside coupling partner used in the synthesis of the provided oligonucleotide has the same structure and / or stereochemical arrangement as any other nucleoside coupling partner of the oligonucleotide. is not it. Exemplary nucleobases and sugars used according to the methods of the invention are described herein. Those skilled in the art of related chemical and synthetic techniques will recognize that any combination of nucleobases and sugars described herein is intended for use by the methods of the invention.

の構造を有し、Ｂ^ＰＲＯは保護核酸塩基である。いくつかの実施形態では、カップリングパートナーは、

の構造を有し、Ｂ^ＰＲＯは保護核酸塩基である。カップリングパートナーとして例示的なキラルホスホラミダイトを下に示す。

Coupling Steps Exemplary coupling procedures, chiral and condensing reagents used in accordance with the present invention are, among other things, Wada I (JP4348077; WO2005 / 014609; WO2005 / 092909), Wada II (WO2010 / 064146), and Wada. It is outlined in III (WO2012 / 039448). The chiral nucleoside coupling partner used in accordance with the present invention is also referred to herein as "Wada amidite". In some embodiments, the coupling partner is

B ^PRO is a protected nucleobase. In some embodiments, the coupling partner is

B ^PRO is a protected nucleobase. An exemplary chiral phosphoramidite as a coupling partner is shown below.

One of the methods used to synthesize a coupling partner is shown in Scheme II below.
Scheme II Coupling partner synthesis example

In some embodiments, the coupling step comprises reacting the free hydroxyl group of a nucleotide unit of an oligonucleotide with a nucleoside coupling partner under appropriate conditions to cause coupling. In some embodiments, a deblocking step is performed prior to the coupling step. For example, in some embodiments, the 5'hydroxyl group of the growing oligonucleotide is blocked (ie protected) and must then be deblocked in order to react with the nucleoside coupling partner.

After deblocking the appropriate hydroxyl groups of the growing oligonucleotide, the support is washed and dried in preparation for delivery of the solution containing the chiral reagent and the solution containing the activator. In some embodiments, the chiral reagent and activator are delivered simultaneously. In some embodiments, simultaneous delivery involves some chiral reagents in a solution (eg, phosphoramidite solution) and some activators in a solution (eg, CMPT solution), with some polarity such as a nitrile solvent (eg, acetonitrile). Includes feeding into an aprotic solvent.

In some embodiments, the coupling step provides a crude composition in which the chiral phosphite product is present in a diastereomeric excess of> 95%. In some embodiments, the chiral phosphite product is present with a diastereomeric excess of greater than 96%. In some embodiments, the chiral phosphite product is present with a diastereomeric excess of greater than 97%. In some embodiments, the chiral phosphite product is present with a diastereomeric excess of greater than 98%. In some embodiments, the chiral phosphite product is present with a diastereomeric excess of greater than 99%.

Capping Steps The methods provided to make chiral controlled oligonucleotides include capping steps. In some embodiments, the capping step is a single step. In some embodiments, the capping step is two steps. In some embodiments, the capping step is more than two steps.

In some embodiments, the capping step comprises capping the free amine of the chiral auxiliary and capping the residual unreacted 5'hydroxyl group. In some embodiments, the free amine of the chiral auxiliary and the unreacted 5'hydroxyl group are capped with the same capping group. In some embodiments, the free amine of the chiral auxiliary and the unreacted 5'hydroxyl group are capped with the same capping group. In some embodiments, the free amine of the chiral auxiliary and the unreacted 5'hydroxyl group are capped with different capping groups. In certain embodiments, capping with different capping groups allows one capping group to be selectively removed prior to the other capping groups during the synthesis of the oligonucleotide. In some embodiments, capping of both groups occurs simultaneously. In some embodiments, capping of both groups occurs repeatedly.

In certain embodiments, capping occurs repeatedly, comprising a first step of capping the free amine and then a second step of capping the free 5'hydroxyl group, both the free amine and the 5'hydroxyl group being the same capping. Capped on the basis. For example, in some embodiments, the free amine of the chiral auxiliary is capped with the same anhydride before the 5'hydroxyl group is capped with the anhydride (eg, phenoxyacetic acid anhydride, ie _Pac2O ). Will be done. In certain embodiments, capping of 5'hydroxyl groups with the same anhydride occurs under different conditions (eg, in the presence of one or more additional reagents). In some embodiments, capping of the 5'hydroxyl group occurs in the presence of an amine base in an ethereal solvent (eg, NMI (N-methylimidazole) in THF). The expression "capping group" is used herein interchangeably with the phrases "protecting group" and "blocking group".

In some embodiments, the amine capping group is characterized by effectively capping the amine to prevent rearrangement and / or degradation of the intermediate phosphite ester species. In some embodiments, the capping group is selected for its ability to protect the amine of the chiral auxiliary with the aim of preventing intramolecular cleavage of the internucleotide-bound phosphorus.

In some embodiments, "shortmers", eg, "nm" (m and n are integers, m <n; n is the number of bases in the target oligonucleotide) with impurities. Therefore, effective capping of hydroxyl groups to prevent the generation of impurities, which results from the reaction of oligonucleotide chains that do not react in the first cycle and react in the next one or more cycles, is 5'hydroxyl. The base capping group is characteristic. The presence of such short mer, especially "n-1], has a detrimental effect on the purity of the crude oligonucleotide, making the final purification of the oligonucleotide time consuming and generally low yield.

In some embodiments, a particular cap is selected based on a tendency to facilitate a particular type of reaction under certain conditions. For example, in some embodiments, the capping group is selected for its ability to facilitate the E1 elimination reaction, which cleaves the cap and / or co-group from the growing oligonucleotide. In some embodiments, the capping group is selected for its ability to facilitate the E2 elimination reaction, which cleaves the cap and / or co-group from the growing oligonucleotide. In some embodiments, the capping group is selected for its ability to facilitate the β-elimination reaction, which cleaves the cap and / or co-group from the growing oligonucleotide.

Modification Steps As used herein, the phrases "modifying step,""modificationstep," and "P-modification step" are used interchangeably to introduce modified nucleotide-to-nucleotide binding. Generally refers to any one or more steps used to do so. In some embodiments, modified internucleotide linkages having the structure of formula I. The P-modification step of the present invention occurs not after the construction of the provided oligonucleotide is completed, but during the construction of the provided oligonucleotide. Thus, each nucleotide unit of the provided oligonucleotide can be individually modified with bound phosphorus during the cycle in which the nucleotide unit is introduced.

In some embodiments, suitable P-modifying reagents are sulfur electrophiles, selenium electrophiles, oxygen electrophiles, boring reagents, or azide reagents.

For example, in some embodiments, the selenium reagent is an elemental selenium, a selenium salt, or a substituted diselenide. In some embodiments, the oxygen electrophile is an elemental oxygen, peroxide, or substituted peroxide. In some embodiments, the borane reagent is borane-amine (eg, N, N-diisopropylethylamine (BH ₃ · DIPEA), borane-pyridine (BH ₃ · Py), borane-2-chloropyridine (BH ₃ ). CPy), borane-aniline (BH ₃ · An)), borane-ether reagent (eg borane-tetratetra (BH ₃ · THF)), borane-dialkyl sulfide reagent (eg BH ₃ · Me ₂ S), aniline -Cyanoborane, or triphenylphosphine-carboalkoxyborane. In some embodiments, the azide reagent comprises an azide group capable of providing an amine group via subsequent reduction.

In some embodiments, the P-modifying reagent is the sulfurizing reagent described herein. In some embodiments, the modification step comprises sulfurizing phosphorus to provide a phosphorothioate bond or a phosphorothioate triester bond. In some embodiments, the modification step provides an oligonucleotide having an internucleotide bond of formula I.

In some embodiments, the invention provides a sulfidation reagent, a method of making the reagent, and a method of using the reagent.

Ｓ－Ｉ、
式中、
Ｒ^ｓ１はＲであり；
Ｒ、Ｌ、及びＲ^１のそれぞれは互いに独立して上及び本明細書に定義され記載される通りである。 In some embodiments, the sulfurizing reagent is a thiosulfonic acid reagent. In some embodiments, the thiosulfonic acid reagent has the structure of formula SI:

SI,
During the ceremony
R ^s1 is R;
R, L, and R ¹ are each independently defined above and as described herein.

Ｓ－ＩＩ、
式中、Ｒ^ｓ１及びＬのそれぞれは互いに独立して上及び本明細書に定義され記載される通りである。 In some embodiments, the sulfurizing reagent is a bis (thiosulfonic acid) reagent. In some embodiments, the bis (thiosulfonic acid) reagent has the structure of formula S-II:

S-II,
In the formula, R ^s1 and L, respectively, are independent of each other and as defined and described herein.

Generally, as defined above, R ^s1 is R, where R is as defined and described above and herein. In some embodiments, R ^s1 is an aliphatic, aryl, heterocyclyl, or heteroaryl that may be substituted. In some embodiments, R ^s1 is an alkyl that may be substituted. In some embodiments, R ^s1 is an alkyl that may be substituted. In some embodiments, R ^s1 is methyl. In some embodiments, R ^s1 is cyanomethyl. In some embodiments, R ^s1 is nitromethyl. In some embodiments, R ^s1 is an aryl that may be substituted. In some embodiments, R ^s1 is a optionally substituted phenyl. In some embodiments, R ^s1 is phenyl. In some embodiments, R ^s1 is p-nitrophenyl. In some embodiments, R ^s1 is p-methylphenyl. In some embodiments, R ^s1 is p-chlorophenyl. In some embodiments, R ^s1 is o-chlorophenyl. In some embodiments, R ^s1 is 2,4,6-trichlorophenyl. In some embodiments, R ^s1 is pentafluorophenyl. In some embodiments, R ^s1 is a substituted heterocyclyl that may be. In some embodiments, R ^s1 is a substituted heteroaryl which may be.

（ＭＴＳ）
である。いくつかの実施形態では、 Ｒ^ｓ１－Ｓ（Ｏ）_２Ｓ－は

（ＴＴＳ）
である。いくつかの実施形態では、 Ｒ^ｓ１－Ｓ（Ｏ）_２Ｓ－は

（ＮＯ_２ＰｈｅＴＳ）
である。いくつかの実施形態では、 Ｒ^ｓ１－Ｓ（Ｏ）_２Ｓ－は

（ｐ－ＣｌＰｈｅＴＳ）
である。いくつかの実施形態では、 Ｒ^ｓ１－Ｓ（Ｏ）_２Ｓ－は

（ｏ－ＣｌＰｈｅＴＳ）
である。いくつかの実施形態では、 Ｒ^ｓ１－Ｓ（Ｏ）_２Ｓ－は

（２，４，６－ＴｒｉＣｌＰｈｅＴＳ）
である。いくつかの実施形態では、 Ｒ^ｓ１－Ｓ（Ｏ）_２Ｓ－は

（ＰｈｅＴＳ）
である。いくつかの実施形態では、 Ｒ^ｓ１－Ｓ（Ｏ）_２Ｓ－は

（ＰＦＰｈｅＴＳ）
である。いくつかの実施形態では、 Ｒ^ｓ１－Ｓ（Ｏ）_２Ｓ－は

（ａ－ＣＮＭＴＳ）
である。いくつかの実施形態では、 Ｒ^ｓ１－Ｓ（Ｏ）_２Ｓ－は

（ａ－ＮＯ_２ＭＴＳ）
である。いくつかの実施形態では、 Ｒ^ｓ１－Ｓ（Ｏ）_２Ｓ－は

（ａ－ＣＦ_３ＭＴＳ）
である。いくつかの実施形態では、 Ｒ^ｓ１－Ｓ（Ｏ）_２Ｓ－は

（ａ－ＣＦ_３ＴＳ）
である。いくつかの実施形態では、 Ｒ^ｓ１－Ｓ（Ｏ）_２Ｓ－は

（ａ－ＣＨＦ_２ＴＳ）
である。いくつかの実施形態では、 Ｒ^ｓ１－Ｓ（Ｏ）_２Ｓ－は

（ａ－ＣＨ_２ＦＴＳ）
である。 In some embodiments, R ^s1 -S (O) ₂ S-

(MTS)
Is. In some embodiments, R ^s1 -S (O) ₂ S-

(TTS)
Is. In some embodiments, R ^s1 -S (O) ₂ S-

(NO ₂ PheTS)
Is. In some embodiments, R ^s1 -S (O) ₂ S-

(P-ClPheTS)
Is. In some embodiments, R ^s1 -S (O) ₂ S-

(O-ClPheTS)
Is. In some embodiments, R ^s1 -S (O) ₂ S-

(2,4,6-TriClPheTS)
Is. In some embodiments, R ^s1 -S (O) ₂ S-

(PheTS)
Is. In some embodiments, R ^s1 -S (O) ₂ S-

(PFPheTS)
Is. In some embodiments, R ^s1 -S (O) ₂ S-

(A-CNMTS)
Is. In some embodiments, R ^s1 -S (O) ₂ S-

(A-NO ₂ MTS)
Is. In some embodiments, R ^s1 -S (O) ₂ S-

(A-CF ₃ MTS)
Is. In some embodiments, R ^s1 -S (O) ₂ S-

(A-CF ₃ TS)
Is. In some embodiments, R ^s1 -S (O) ₂ S-

(A-CHF ₂ TS)
Is. In some embodiments, R ^s1 -S (O) ₂ S-

(A-CH ₂ FTS)
Is.

In some embodiments, the sulfurizing reagent has a structure of SI or S-II, where L is —S—R ^L3- or —SC (O) ^-RL3- . In some embodiments, L is —S—R ^L3- or —SC (O) ^-RL3- and ^RL3 is a C ₁ to C ₆ alkylene that may be substituted. In some embodiments, L is -S-R ^L3- or -SC (O) -R ^L3- and RL ³ is C ₁ to C ₆ alkenylene which may be substituted. In some embodiments, L is -S-R ^L3- or -SC (O) -R ^L3- and ^RL3 is a C ₁ to C ₆ alkylene that may be substituted, one or more. The plurality of methylene units may be substituted independently of _each other by C1 to C6 _alkenylene , arylene, or heteroarylene which may be substituted. In some embodiments, in some embodiments, ^RL3 may be substituted-S- (C ₁ to C ₆ alkenylene)-,-S- (C ₁ to C ₆ alkylene)-,-. S- (C ₁ to C ₆ alkylene) -allylen- (C ₁ to C ₆ alkylene)-, -S-CO-allylene- (C ₁ to C ₆ alkylene)-or -S-CO- (C ₁ to C ₆ alkylene) -allylene- (C ₁ to C ₆ alkylene)-. In some embodiments, the sulfurizing reagent has a structure of SI or S-II, where L is -S-R ^L3- or -SC (O) ^-RL3- and the sulfur atom is R. Combine to ¹ .

In some embodiments, the sulfurizing reagent has a structure of SI or S-II, where L is alkylene, alkenylene, arylene, or heteroarylene.

である。いくつかの実施形態では、Ｌは

であり、イオウ原子はＲ^１に結合する。 In some embodiments, the sulfurizing reagent has a structure of SI or S-II, where L is

Is. In some embodiments, L is

And the sulfur atom is bonded to ^R1 .

である。いくつかの実施形態では、Ｒ^１は

であり、イオウ原子はＬに結合する。 In some embodiments, the sulfurizing reagent has a structure of SI or S-II, where ^R1 is

Is. In some embodiments, R ¹ is

And the sulfur atom is bonded to L.

であり、イオウ原子はＲ^１に結合し、Ｒ１は

であり、イオウ原子はＬに結合する。 In some embodiments, the sulfurizing reagent has a structure of SI or S-II, where L is

The sulfur atom is bonded to ^R1 and R1 is

And the sulfur atom is bonded to L.

In some embodiments, the sulfurizing reagent has a structure of SI or S-II, R ¹ is —S—R ^L2 , and ^RL2 is as defined and described herein above. be. In some embodiments, ^RL2 is -S- (C ₁ to C ₆ alkylene) -heterocyclyl, -S- (C ₁ to C ₆ alkenylene) -heterocyclyl, -S- (C ₁ to C ₆ alkylene)-. Substituted groups selected from N (R') ₂ , -S- (C ₁ to C ₆ alkylene) -N (R') ₃ , each R'defined above and herein. As described in.

In some embodiments, -L-R ¹ is -R ^L3 -S-S-R ^L2 , where the variables are defined above independently of each other and as described herein. In some embodiments, -L-R ¹ is -R ^L3 -C (O) -S-S-R ^L2 , where the variables are defined above independently of each other and as described herein. Is.

An exemplary bis (thiosulfonic acid) reagent of formula S-II is shown below:

In some embodiments, the sulfurizing reagent is a compound having one of the following formulas:
S ₈ , R ^s2 -S-S-R ^s3 , or R ^s2 -SX ^s -R ^s3 ,
During the ceremony
Each of R ^s2 and R ^s3 is independent of each other and is aliphatic, aminoalkyl, carbocyclyl, heterocyclyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide, or thio. Substituted groups selected from the carbonyl; or R ^s2 and R ^s3 together with the atoms to which they are attached form a optionally substituted heterocyclic or heteroaryl ring. ;
^Xs is -S (O) _2- , -O-, or -N (R')-; and R'is as defined and described above and herein.

である。いくつかの実施形態では、硫化試薬はＳ_８、

である。いくつかの実施形態では、硫化試薬は

である。 In some embodiments, the sulfurizing reagent is _S8 ,

Is. In some embodiments, the sulfurizing reagent is _S8 ,

Is. In some embodiments, the sulfurizing reagent

Is.

Exemplary sulfurizing reagents are shown in Table 5 below.

Table 5 Exemplary Sulfurization Reagents

式中、Ｒ^Ｓ１、Ｌ、及びＲ^１のそれぞれは上及び本明細書に記載され定義される通りである。 In some embodiments, the provided sulfurizing reagents are used to modify H-phosphonates. For example, in some embodiments, the H-phosphonate oligonucleotide is synthesized, for example, using the method of Wada I or Wada II and modified with the sulfide reagent of formula SI or S-II:

In the formula, ^RS1 , L, and ^R1 are as described above and defined herein, respectively.

（式中、Ｗ、Ｙ、及びＺのそれぞれは上及び本明細書に記載され定義される通りであり、シリル化試薬とともにシリルオキシホスホネートを提供する）のＨ－ホスホネートを反応させるステップと、
ｉｉ）シリルオキシホスホネートを構造Ｓ－Ｉ又はＳ－ＩＩ：

の硫化試薬と反応させ、ホスホロチオトリエステルを提供するステップと、
を含む。 In some embodiments, the invention provides a method of synthesizing a phosphorothioate triester, which method is:
i) Structure:

The step of reacting with H-phosphonate (each of W, Y, and Z in the formula is as described above and defined herein and provides silyloxyphosphonate with silylation reagent).
ii) Structure SI or S-II: Cyriloxyphosphonate:

To provide a phosphorothiotriester by reacting with a sulfide reagent of
including.

In some embodiments, selenium electrophiles are used in place of sulfurizing reagents to introduce modifications to internucleotide bonds. In some embodiments, the selenium electrophile is a compound having one of the following formulas:
Se, R ^s2 -Se-Se-R ^s3 , or R ^s2 -Se-X ^s -R ^s3 ,
In the formula, each of R ^s2 and R ^s3 is independent of each other, aliphatic, aminoalkyl, carbocyclyl, heterocyclyl, heterocycloalkyl, aryl, heteroaryl, alkyloxy, aryloxy, heteroaryloxy, acyl, amide, imide. , Or a optionally substituted group selected from thiocarbonyl; or R ^s2 and R ^s3 together with the atom to which they are attached may be substituted heterocyclic or heteroaryl rings. Form;
^Xs is -S (O) _2- , -O-, or -N (R')-; and R'is as defined and described above and herein.

の化合物である。いくつかの実施形態では、セレニウム求電子試薬はＳｅ又は

である。 In other embodiments, the selenium electrophile is Se, KSeCN,

It is a compound of. In some embodiments, the selenium electrophile is Se or

Is.

In some embodiments, the sulfurizing reagents used according to the invention are substituted sulfur (eg, -SR) rather than a single sulfur atom (eg, ^—S— or = S), where the component that moves to phosphorus during sulfurization is not a single sulfur atom (eg, —S— or = S). It is characterized by being.

In some embodiments, the sulfurizing reagent used according to the invention is characterized in that the activity of the reagent can be regulated by modifying the reagent with a predetermined electron-withdrawing group or electron donating group.

In some embodiments, the sulfurizing reagent used according to the present invention is characterized in that it is crystalline. In some embodiments, the sulfurizing reagent used according to the present invention is characterized in that it has a high degree of crystallinity. In certain embodiments, the sulfurizing reagents used in accordance with the present invention are characterized by the ease of purification of the reagents, for example by recrystallization. In certain embodiments, the sulfurizing reagents used in accordance with the present invention are characterized in that they are substantially free of sulfur-containing impurities. In some embodiments, sulfurizing reagents that are substantially free of sulfur-containing impurities show improved efficiency.

In some embodiments, the chiral control oligonucleotide provided comprises one or more phosphate diester bonds. To synthesize such chiral controlled oligonucleotides, one or more modification steps may be replaced with oxidation steps to introduce equivalent phosphodiester bonds. In some embodiments, the oxidation step is carried out in a similar manner to conventional oligonucleotide synthesis. In some embodiments, the oxidation step comprises the use of I ₂ . In some embodiments, the oxidation step comprises the use of I ₂ and pyridine. In some embodiments, the oxidation step comprises the use of 0.02 MI ₂ in a THF / pyridine / water (70: 20: 10-v / v / v) co-solvent system. An exemplary cycle is shown in Scheme IC.

である。いくつかの実施形態では、

は、サイクル終了後、標準脱保護／放出手順の間、ホスホロチオエートジエステル結合へと変換される。例をさらに下に示す。 In some embodiments, a phosphorothioate precursor is used to synthesize a chiral-controlled oligonucleotide containing a phosphorothioate bond. In some embodiments, such phosphorothioate precursors are

Is. In some embodiments,

Is converted to phosphodiester bonds after the end of the cycle during standard deprotection / release procedures. An example is shown below.

である。当業者に理解されるように、合成の間、ホスホロチオエート前駆体、例えば、

を使用する利点の１つは、

はある特定の条件下でホスホロチオエートより安定しているということである。 In some embodiments, the provided chiral control oligonucleotide comprises one or more phosphate diester bonds and one or more phosphodiester bonds. In some embodiments, the provided chiral-controlled oligonucleotide comprises one or more phosphate diester bonds and one or more phosphodiester bonds, and at least one phosphate diester bond is 3'to 5'. When synthesized into, it is introduced after all phosphodiester bonds. To synthesize such chiral controlled oligonucleotides, in some embodiments, one or more modification steps may be replaced with an oxidation step to introduce an equivalent phosphodiester bond, or the phosphorothioate precursor is phosphorothioate. Introduce for each of the diester bonds. In some embodiments, the phosphorothioate precursor is converted to a phosphodiester bond after achieving the desired oligonucleotide length. In some embodiments, the phosphorothioate precursor is converted to a phosphodiester bond during or after the end of the cycle in a deprotection / release step. In some embodiments, the phosphorothioate precursor is characterized in that it can be removed by the β-elimination pathway. In some embodiments, the phosphorothioate precursor is

Is. As will be appreciated by those of skill in the art, phosphorothioate precursors during synthesis, eg,

One of the advantages of using

Is more stable than phosphorothioate under certain conditions.

In some embodiments, the phosphorothioate precursor is a phosphorus protecting group described herein, eg, 2-cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, Benzyl, o-nitrobenzyl, 2- (p-nitrophenyl) ethyl (NPE or Npe), 2-phenylethyl, 3- (N-tert-butylcarboxamide) -1-propyl, 4-oxopentyl, 4-methylthio -1-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3- (2-pyridyl) -1-propyl, 2- [N-methyl-N- (2-pyridyl) ] Aminoethyl, 2- (N-formyl, N-methyl) aminoethyl, 4- [N-methyl-N- (2,2,2-trifluoroacetyl) amino] butyl. An example is shown below.

Methods for synthesizing the desired sulfide reagent are described herein and in the Examples.

As noted above, in some embodiments, sulfurization occurs under conditions that cleave the chiral reagent from the growing oligonucleotide. In some embodiments, sulfurization occurs under conditions that do not cleave the chiral reagent from the growing oligonucleotide.

In some embodiments, the sulfurizing reagent is dissolved in a suitable solvent and delivered to the column. In certain embodiments, the solvent is a polar aprotic solvent such as a nitrile solvent. In some embodiments, the solvent is acetonitrile. In some embodiments, the solution of the sulfide reagent is in a nitrile solvent (eg, acetonitrile) with the sulfide reagent (eg, the thiosulfonic acid derivative described herein) in BSTFA (N, O-bis-trimethylsilyl-). Prepared by mixing with trifluoroacetoamide). In some embodiments, BSTFA is not included. For example, the inventor has found that a relatively more reactive sulfidation reagent of the general formula R ^s2 -SS (O) ₂ -R ^s3 can often be successfully involved in the sulfidation reaction in the absence of BSTFA. rice field. To give just one example, the inventor demonstrated that BSTFA is not needed if R ^s2 is p-nitrophenyl and R ^s3 is methyl. With reference to this disclosure, one of ordinary skill in the art will be readily able to determine other situations and / or sulfurizing reagents that do not require BSTFA.

In some embodiments, the sulfurization step is performed at room temperature. In some embodiments, the sulfurization step is performed at a lower temperature, such as about 0 ° C, about 5 ° C, about 10 ° C, or about 15 ° C. In some embodiments, the sulfurization step is performed at a temperature above about 20 ° C.

In some embodiments, the sulfurization reaction is carried out for about 1 minute to about 120 minutes. In some embodiments, the sulfurization reaction is carried out for about 1 minute to about 90 minutes. In some embodiments, the sulfurization reaction is carried out for about 1 minute to about 60 minutes. In some embodiments, the sulfurization reaction is carried out for about 1 minute to about 30 minutes. In some embodiments, the sulfurization reaction is carried out for about 1 minute to about 25 minutes. In some embodiments, the sulfurization reaction is carried out for about 1 minute to about 20 minutes. In some embodiments, the sulfurization reaction is carried out for about 1 minute to about 15 minutes. In some embodiments, the sulfurization reaction is carried out for about 1 minute to about 10 minutes. In some embodiments, the sulfurization reaction is carried out for about 5 to about 60 minutes.

In some embodiments, the sulfurization reaction is carried out for about 5 minutes. In some embodiments, the sulfurization reaction is carried out for about 10 minutes. In some embodiments, the sulfurization reaction is carried out for about 15 minutes. In some embodiments, the sulfurization reaction is carried out for about 20 minutes. In some embodiments, the sulfurization reaction is carried out for about 25 minutes. In some embodiments, the sulfurization reaction is carried out for about 30 minutes. In some embodiments, the sulfurization reaction is carried out for about 35 minutes. In some embodiments, the sulfurization reaction is carried out for about 40 minutes. In some embodiments, the sulfurization reaction is carried out for about 45 minutes. In some embodiments, the sulfurization reaction is carried out for about 50 minutes. In some embodiments, the sulfurization reaction is carried out for about 55 minutes. In some embodiments, the sulfurization reaction is carried out for about 60 minutes.

It was unexpectedly found that some of the sulfurization modification products produced according to the method of the present invention are unexpectedly stable. In some embodiments, the unexpectedly stable product is a phosphorothioate triester. In some embodiments, the unexpectedly stable product is a chiral controlled oligonucleotide containing one or more internucleotide linkages having the structure of formula Ic.

Those skilled in the art will appreciate that the sulfurization methods described herein and the sulfurization reagents described herein are also related to modified H-phosphonate oligonucleotides, such as those described in Wada II (WO2010 / 064146). Recognize that it is useful.

In some embodiments, the sulfurization reaction has a stepwise sulfurization efficiency of at least about 80%, 85%, 90%, 95%, 96%, 97%, or 98%. In some embodiments, the sulfurization reaction provides a crude dinucleotide product composition with a purity of at least 98%. In some embodiments, the sulfurization reaction provides a crude tetranucleotide product composition with a purity of at least 90%. In some embodiments, the sulfurization reaction provides a crude dodecanucleotide product composition with a purity of at least 70%. In some embodiments, the sulfidation reaction provides a crude icosabinucleotide product composition with a purity of at least 50%.

Upon completion of the step of modifying bound phosphorus, the oligonucleotide goes through another deblocking step in preparation for resuming the cycle. In some embodiments, the chiral auxiliary remains intact after sulfidation and is deblocked during the subsequent deblocking step, which always occurs before resuming the cycle. The steps of deblocking, coupling, capping, and modification are repeated until the growing oligonucleotide reaches the desired length, at which point the oligonucleotide is immediately cleaved from the solid support. Either it can be left attached to the support for purification purposes and then cut later. In some embodiments, one or more protecting groups are present at one or more of the nucleotide bases, and cleavage of the oligonucleotide from the support and deprotection of the base occurs in a single step. In some embodiments, one or more protecting groups are present at one or more of the nucleotide bases, and cleavage of the oligonucleotide from the support and deprotection of the base occurs in more than one step. In some embodiments, deprotection and cleavage from the support occur, for example, under basic conditions with one or more amine bases. In certain embodiments, the amine base may include propylamine. In certain embodiments, the amine base may include pyridine.

In some embodiments, cutting and / or deprotection from the support occurs at high temperatures of about 30 ° C to about 90 ° C. In some embodiments, cutting and / or deprotection from the support occurs at high temperatures of about 40 ° C to about 80 ° C. In some embodiments, cutting and / or deprotection from the support occurs at high temperatures of about 50 ° C to about 70 ° C. In some embodiments, cutting and / or deprotection from the support occurs at a high temperature of about 60 ° C. In some embodiments, cutting and / or deprotection from the support occurs at ambient temperature.

Exemplary purification procedures are described herein and are well known in the art, or either.

Of note, it is advantageous to remove chiral auxiliary from the growing oligonucleotide during each cycle for at least the following reasons:
(1) At the end of oligonucleotide synthesis to introduce potentially sensitive functional groups into phosphorus, it is not necessary to remove the co-groups in any separation step, (2) prone to side reactions and / or subsequent The avoidance of unstable phosphorus-auxiliary group intermediates that are prone to interfere with chemical action. In this way, the removal of chiral auxiliary during each cycle makes the whole synthesis more efficient.

The deblocking step is described above in the context of the cycle, while additional general methods are included as below.

Deblocking Step In some embodiments, the coupling step is preceded by the deblocking step. For example, in some embodiments, the 5'hydroxyl group of the growing oligonucleotide must be blocked (ie protected) and subsequently deblocked for reaction with the nucleoside coupling partner. It doesn't become.

より選択され、式中、Ｒ^ａ１及びＲ^ａ２のそれぞれは互いに独立して水素又は置換されていてもよいアルキル若しくはアリールであり、Ｒ^ａ３は置換されていてもよいアルキル又はアリールである。 In some embodiments, acidification is used to remove the blocking groups. In some embodiments, the acid is a Bronsted acid or a Lewis acid. A useful Bronsted acid is a carboxylic acid having a pKa (25 ° C. in water) value of -0.6 (trifluoroacetic acid) to 4.76 (acetic acid) in an organic solvent or water (in the case of 80% acetic acid). , Alkylsulfonic acid, arylsulfonic acid, phosphoric acid and its derivatives, phosphonic acid and its derivatives, alkylphosphonic acid and its derivatives, arylphosphonic acid and its derivatives, phosphinic acid, dialkylphosphinic acid, and diarylphosphinic acid. .. The concentration of acid (1-80%) used in the acidification step depends on the acidity of the acid. Strongly acidic conditions result in depurine / depyrimidine, and the acid strength must be taken into account as the prynyl base or pyrimidinyl base is cleaved from the ribose ring and / or other sugar rings. In some embodiments, the acid is R ^a1 COOH, R ^a1 SO ₃ H, R ^a3 SO ₃ H,

More selected, in the formula, R ^a1 and R ^a2 are each independently hydrogen or optionally substituted alkyl or aryl, and R ^a3 is optionally substituted alkyl or aryl.

In some embodiments, acidification is achieved by Lewis acid in an organic solvent. An exemplary such useful Lewis acid is Zn (X ^a ) ₂ , where X ^a is Cl, Br, I, or CF ₃ SO ₃ .

In some embodiments, the acidification step comprises adding an effective amount of Bronsted acid or Lewis acid to remove the blocking group without removing the purine component from the condensation intermediate.

Acids useful for the acidification step are also 10% phosphoric acid in organic solvent, 10% hydrochloric acid in organic solvent, 1% trifluoroacetic acid in organic solvent, 3% dichloroacetic acid or trichloroacetic acid in organic solvent or 80% acetic acid in water. However, it is not limited to these. The concentration of any Bronsted or Lewis acid used in this step is chosen so that the concentration of the acid does not exceed the concentration that causes cleavage of the nucleobase from the sugar component.

In some embodiments, acidification comprises adding about 1% trifluoroacetic acid in an organic solvent. In some embodiments, acidification comprises adding about 0.1% to about 8% trifluoroacetic acid in an organic solvent. In some embodiments, acidification comprises adding 3% dichloroacetic acid or trichloroacetic acid in an organic solvent. In some embodiments, acidification comprises adding about 0.1% to about 10% dichloroacetic acid or trichloroacetic acid in an organic solvent. In some embodiments, acidification comprises adding 3% trichloroacetic acid in an organic solvent. In some embodiments, acidification comprises adding about 0.1% to about 10% trichloroacetic acid in an organic solvent. In some embodiments, acidification comprises adding 80% acetic acid in water. In some embodiments, acidification is about 50% to about 90%, or about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 70% to about 70% in water. Includes the addition of 90% acetic acid. In some embodiments, acidification further comprises adding a cationic scavenger to an acidic solvent. In certain embodiments, the cationic scavenger may be triethylsilane or triisopropylsilane. In some embodiments, the blocking group is deblocked by acidification, which comprises adding 1% trifluoroacetic acid in an organic solvent. In some embodiments, the blocking group is deblocked by acidification, which comprises adding 3% dichloroacetic acid in an organic solvent. In some embodiments, the blocking group is deblocked by acidification, which comprises adding 3% trichloroacetic acid in an organic solvent. In some embodiments, the blocking group is deblocked by acidification, which comprises adding 3% trichloroacetic acid in dichloromethane.

In certain embodiments, the method of the invention is completed on a synthesizer and the step of deblocking the hydroxyl groups of the growing oligonucleotide comprises delivering some amount of solvent to the synthesizer column. , This column contains a solid support to which oligonucleotides are attached. In some embodiments, the solvent is a halogenated solvent (eg, dichloromethane). In certain embodiments, the solvent comprises some amount of acid. In some embodiments, the solvent comprises some amount of organic acid, such as trichloroacetic acid. In certain embodiments, the acid is present in an amount of about 1% to about 20% w / v. In certain embodiments, the acid is present in an amount of about 1% to about 10% w / v. In certain embodiments, the acid is present in an amount of about 1% to about 5% w / v. In certain embodiments, the acid is present in an amount of about 1% to about 3% w / v. In certain embodiments, the acid is present in an amount of about 3% w / v. Methods for deblocking hydroxyl groups are further described herein. In some embodiments, the acid is present at 3% w / v in dichloromethane.

In some embodiments, the chiral auxiliary is removed prior to the deblocking step. In some embodiments, the chiral auxiliary is removed during the deblocking step.

In some embodiments, the end of the cycle is performed prior to the deblocking step. In some embodiments, the end of the cycle is performed after the deblocking step.

General Conditions for Removal of Blocking / Protecting Groups Functional groups such as hydroxyl or amino components located on nucleobases or sugars are routinely blocked by blocking (protecting) groups (components) during synthesis and then removed. Be blocked. In general, blocking groups inactivate the chemical functional components of the molecule for specific reaction conditions and later remove the blocking groups from such functional components within the molecule with virtually no damage to the rest of the molecule. It is possible (see, eg, Green and Wuts, Protective Groups in Organic Synthesis, 2nd Ed., John Willey & Sons, New York, 1991). For example, the amino groups are phthalimide, 9-fluorenylmethoxycarbonyl (FMOC), triphenylmethylsulphenyl, t-BOC, 4,4'-dimethoxytrityl (DMTr), 4-methoxytrityl (MMTr). , 9-Phenylxanthin-9-yl (Pixil), Trityl (Tr), or 9- (p-methoxyphenyl) xanthin-9-yl (MOX) can be blocked with a nitrogen blocking group. The carboxyl group can be protected as an acetyl group. The hydroxy groups are tetrahydropyranyl (THP), t-butyldimethylsilyl (TBDMS), 1-[(2-chloro-4-methyl) phenyl] -4-methoxypiperidin-4-yl (Ctp), 1-( 2-Fluorophenyl) -4-methoxypiperidine-4-yl (Fpmp), 1- (2-chloroethoxy) ethyl, 3-methoxy-1,5-dicarbomethoxypentane-3-yl (MDP), bis ( 2-acetoxyethoxy) methyl (ACE), triisopropylsilyloxymethyl (TOM), 1- (2-cyanoethoxy) ethyl (CEE), 2-cyanoethoxymethyl (CEM), [4- (N-dichloroacetyl-) It can be protected as N-methylamino) benzyloxy] methyl, 2-cyanoethyl (CN), pivaloyloxymethyl (PivOM), levunyl oxymethyl (ALE) and the like. Other representative hydroxyl block groups have been described (see, eg, Beaucage et al., Tetrahedron, 1992, 46, 2223). In some embodiments, the hydroxyl block groups are trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthine-9-yl (pixyl) and 9- (p-methoxyphenyl) xanthin-9-yl. It is an acid-unstable group such as (MOX). Chemical functional groups can also be blocked by including them in precursor form. In this way, the azide group is easily converted to an amine and can be considered as a blocking form of the amine. Further representative protecting groups utilized in nucleic acid synthesis are known (see, eg, Arawal et al., Protocols for Oligonucleotide Conjugates, Eds., Humana Press, New Jersey, 1994, Vol. 26, pp. 1-72). ).

Various methods for removing blocking groups from nucleic acids are known and used. In some embodiments, all blocking groups are removed. In some embodiments, some of the blocking groups are removed. In some embodiments, the reaction conditions can be adjusted to selectively remove a given blocking group.

In some embodiments, if a nucleobase blocking group is present, it can be cleaved with an acidic reagent after construction of the provided oligonucleotide. In some embodiments, if a nucleobase blocking group is present, it is not cleaved under acidic or basic conditions, for example with a fluoride salt or a hydrofluoric acid complex. In some embodiments, if a nucleobase blocking group is present, it can be cleaved in the presence of a base or basic solvent after construction of the provided oligonucleotide. In certain embodiments, one or more of the nucleobase blocking groups can be cleaved in the presence of a base or basic solvent after the construction of the provided oligonucleotide, but the construction of the provided oligonucleotide. It is characterized by being stable against certain conditions of one or more deprotection steps that occur in the meantime.

In some embodiments, the nucleobase does not require a blocking group. In some embodiments, the nucleobase requires a blocking group. In some embodiments, a given nucleobase requires one or more blocking groups, while other nucleobases do not require one or more blocking groups.

In some embodiments, the oligonucleotide is cleaved from the solid support after synthesis. In some embodiments, cleavage from the solid support comprises the use of propylamine. In some embodiments, cleavage from the solid support comprises the use of propylamine in pyridine. In some embodiments, cleavage from the solid support comprises the use of 20% propylamine in pyridine. In some embodiments, cleavage from the solid support comprises the use of propylamine in anhydrous pyridine. In some embodiments, cleavage from the solid support comprises the use of 20% propylamine in anhydrous pyridine. In some embodiments, cleavage from the solid support comprises the use of polar aprotic solvents such as acetonitrile, NMP, DMSO, sulfone, and / or lutidine. In some embodiments, cleavage from the solid support is a solvent, such as a polar aprotic solvent, and one or more primary amines (eg, C _1-10 amines), and / or one or more. Includes the use of one or more of methoxylamines, hydrazines, and pure anhydrous ammonia.

In some embodiments, deprotection of oligonucleotides comprises the use of propylamine. In some embodiments, deprotection of oligonucleotides comprises the use of propylamine in pyridine. In some embodiments, deprotection of the oligonucleotide comprises the use of 20% propylamine in pyridine. In some embodiments, deprotection of the oligonucleotide comprises the use of propylamine in anhydrous pyridine. In some embodiments, deprotection of the oligonucleotide comprises the use of 20% propylamine in anhydrous pyridine.

In some embodiments, the oligonucleotide is deprotected during cleavage.

In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed at approximately room temperature. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed at elevated temperature. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is about 30 ° C, 40 ° C, 50 ° C, 60 ° C, 70 ° C, 80 ° C, 90 ° C, or 100 ° C. Perform at a higher temperature than each of the above. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is about 30 ° C, 40 ° C, 50 ° C, 60 ° C, 70 ° C, 80 ° C, 90 ° C, or 100 ° C. It will be carried out at. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed at about 40 ° C-80 ° C. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed at about 50 ° C to 70 ° C. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed at about 60 ° C.

In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is 0.1 hours, 1 hour, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours. , 30 hours, or 40 hours, respectively. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed for about 0.1-5 hours. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed for about 3-10 hours. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed for about 5-15 hours. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed for about 10-20 hours. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed for about 15-25 hours. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed for about 20-40 hours. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed for about 2 hours. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed for about 5 hours. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed for about 10 hours. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed for about 15 hours. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed for about 18 hours. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed for about 24 hours.

In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is 0.1 hours, 1 hour, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours at room temperature. , 24 hours, 30 hours, or 40 hours, respectively. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed at room temperature for about 5 to 48 hours. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed at room temperature for about 10-24 hours. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed at room temperature for about 18 hours. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed at high temperature for 0.1 hours, 1 hour, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours. , 24 hours, 30 hours, or 40 hours, respectively. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed at elevated temperature for about 0.5-5 hours. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed at about 60 ° C. for about 0.5-5 hours. In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, is performed at about 60 ° C. for about 2 hours.

In some embodiments, cleavage of the oligonucleotide from the solid support, or deprotection of the oligonucleotide, comprises the use of propylamine at room temperature or high temperature for 0.1 hours, 1 hour, 2 hours, 5 hours. Perform for 10 hours, 15 hours, 20 hours, 24 hours, 30 hours, or 40 hours, respectively. Exemplary conditions are 20% propylamine in pyridine for about 18 hours at room temperature and 20% propylamine in pyridine for about 18 hours at 60 ° C.

In some embodiments, the invention provides a method of making a chiral controlled oligonucleotide, which method is:
(1) Coupling steps and
(2) Capping step and
(3) Modification step and
(4) Deblocking step and (5) Repeating steps (1) to (4) until the desired length is achieved, and
The chiral control oligonucleotide comprises at least one phosphodiester bond or at least one internucleotide bond having the structure of formula Ic.

In some embodiments, the invention provides a method of making a chiral controlled oligonucleotide, which method is:
(1) Coupling steps and
(2) Capping step and
(3) Modification step and
(4) Deblocking step and (5) Repeating steps (1) to (4) until the desired length is achieved, and
At least one cycle of (1) to (4) forms a phosphodiester bond.

In some embodiments, the invention provides a method of making a chiral controlled oligonucleotide, which method is:
(1) Coupling steps and
(2) Capping step and
(3) Modification step and
(4) Deblocking step and (5) Repeating steps (1) to (4) until the desired length is achieved, and
At least one cycle of (1) to (4) forms an internucleotide bond having the structure of formula Ic.

又は

を含み、式中、Ｂ^ＰＲＯは保護核酸塩基であり；
キャッピングステップはキラル補助基におけるアミノ基のキャッピング及び未反応５’－ＯＨのキャッピングを含み；
修飾ステップは結合リンへの－Ｓ－Ｌ－Ｒ^１基の導入を含み、式中、Ｌ及びＲ^１のそれぞれは互いに独立して上に定義され本明細書に記載される通りであるり；
脱ブロッキング（ｄｅｌｏｃｋｉｎｇ）ステップは酸の使用を含む。 In some embodiments, the invention provides a method of making a chiral controlled oligonucleotide, which method is:
(1) Coupling steps and
(2) Capping step and
(3) Modification step and
(4) Deblocking step and (5) Repeating steps (1) to (4) until the desired length is achieved, and
Including
The coupling step is an activating group and

Or

In the formula, B ^PRO is a protected nucleobase;
Capping steps include capping of amino groups at chiral auxiliary and capping of unreacted 5'-OH;
The modification step involves the introduction of ^one -S-L-R to the bound phosphorus, each of L and R ¹ in the formula being independently defined above and as described herein;
The deblocking step involves the use of acid.

を含み、式中、Ｂ^ＰＲＯは保護核酸塩基であり；
キャッピングステップはキラル補助基におけるアミノ基のキャッピング及び未反応５’－ＯＨのキャッピングを含み；
修飾ステップは結合リンへの－Ｓ－Ｌ－Ｒ^１基の導入を含み、式中、Ｌ及びＲ^１のそれぞれは互いに独立して上に定義され本明細書に記載される通りであるり；
脱ブロッキング（ｄｅｌｏｃｋｉｎｇ）ステップは酸の使用を含む。 In some embodiments, the invention provides a method of making a chiral controlled oligonucleotide, which method is:
(1) Coupling steps and
(2) Capping step and
(3) Modification step and
(4) Deblocking step and (5) Repeating steps (1) to (4) until the desired length is achieved, and
Including
Coupling steps are CMPT and

In the formula, B ^PRO is a protected nucleobase;
Capping steps include capping of amino groups at chiral auxiliary and capping of unreacted 5'-OH;
The modification step involves the introduction of ^one -S-L-R to the bound phosphorus, each of L and R ¹ in the formula being independently defined above and as described herein;
The deblocking step involves the use of acid.

In some embodiments, the activator is a "Wada" activator, i.e., the activator is based on any of the Wada I, II, or III documents cited above.

An exemplary activation group is shown below:

An exemplary cycle is shown in Scheme Ib.
Introduction of Scheme I-b phosphorothioate binding

In some embodiments, the invention provides a method of making a chiral controlled oligonucleotide, which method is:
(1) Coupling steps and
(2) Capping step and
(3) Modification step and
(4) Deblocking step and (5) Repeating steps (1) to (4) until the desired length is achieved, and
Including
Chiral controlled oligonucleotides include at least one phosphodiester bond or at least one internucleotide bond of formula I-c, and at least one phosphate diester nucleotide bond;
At least one modification step is replaced by an oxidation step.

In some embodiments, the invention provides a method of making a chiral controlled oligonucleotide, which method is:
(1) Coupling steps and
(2) Capping step and
(3) Modification step and
(4) Deblocking step and (5) Repeating steps (1) to (4) until the desired length is achieved, and
Including
Chiral controlled oligonucleotides include at least one phosphodiester bond or at least one internucleotide bond of formula I-c, and at least one phosphate diester nucleotide bond;
At least one modification step is replaced by an oxidation step involving the use of I ₂ .

An exemplary cycle is shown in Scheme IC.
Introduction of both phosphate diesters and modified internucleotide bonds in Scheme I-c chiral controlled oligonucleotides

In Scheme I-c, oligonucleotides (or nucleotides, or oligonucleotides with modified internucleotide linkages) on the solid support (C-1) are coupled with phosphoramidite C-2. After coupling and capping, an oxidation step is performed. After deblocking, a phosphate diester bond is formed. The cycle product C-3 either resumes cycle C to introduce more phosphodiester bonds, initiates another cycle to introduce other types of internucleotide bonds, or proceeds to the end of the cycle. It will be either.

の構造を有する。 In some embodiments, non-chiral pure phosphoramidite can be used in place of C-2 in Scheme Ic. In some embodiments, DMTr-protected β-cyanoethyl phosphoramidite is used. In some embodiments, the phosphoramidite used is

Has the structure of.

In some embodiments, the invention provides a method of making a chiral controlled oligonucleotide, which method is:
(1) Coupling steps and
(2) Capping step and
(3) Modification step and
(4) Deblocking step and (5) Repeating steps (1) to (4) until the desired length is achieved, and
Including
Chiral controlled oligonucleotides contain one or more phosphodiester bonds;
One or more phosphodiester precursors are formed for each of the phosphodiester bonds.

In some embodiments, the invention provides a method of making a chiral controlled oligonucleotide, which method is:
(1) Coupling steps and
(2) Capping step and
(3) Modification step and
(4) Deblocking step and (5) Repeating steps (1) to (4) until the desired length is achieved, and
Including
Chiral controlled oligonucleotides contain at least one phosphodiester bond;
One or more phosphodiester precursors are formed for each of the phosphodiester bonds;
Each phosphodiester precursor is converted to a phosphodiester bond after the desired length has been achieved.

In some embodiments, the invention provides a method of making a chiral controlled oligonucleotide, which method is:
(1) Coupling steps and
(2) Capping step and
(3) Modification step and
(4) Deblocking step and (5) Repeating steps (1) to (4) until the desired length is achieved, and
Including
Chiral controlled oligonucleotides include at least one phosphodiester bond and at least one phosphate diester nucleotide bond;
At least one modification step is replaced by an oxidation step;
At least one modification step is performed to introduce a phosphodiester precursor to become each of the phosphodiester bonds;
Each phosphodiester precursor is converted to a phosphodiester bond after the desired length has been achieved.

In some embodiments, the use of a phosphodiester precursor increases the stability of the oligonucleotide during synthesis. In some embodiments, the use of phosphodiester precursors improves the efficiency of chiral controlled oligonucleotide synthesis. In some embodiments, the use of phosphodiester precursors improves the yield of chiral controlled oligonucleotide synthesis. In some embodiments, the use of phosphodiester precursor precursors improves the product purity of chiral controlled oligonucleotide synthesis.

である。いくつかの実施形態では、

は、脱保護／放出の間、ホスホロチオエートジエステル結合に変換される。例示的なサイクルをスキームＩ－ｄに示す。さらに例を下に示す。
スキームＩ－ｄ キラル制御オリゴヌクレオチド合成におけるホスホロチオエートジエステル前駆体

In some embodiments, the phosphodiester precursor in the above method is

Is. In some embodiments,

Is converted to a phosphodiester bond during deprotection / release. An exemplary cycle is shown in Scheme Id. Further examples are shown below.
Scheme I-d Phosphodiester precursor in chiral controlled oligonucleotide synthesis

が、ホスホロチオエートジエステル結合の代わりに導入される。いくつかの実施形態では、かかる置き換えは、所定のステップ、例えば酸化ステップの間、合成効率の向上を提供した。いくつかの実施形態では、ホスホロチオエートジエステル前駆体の使用は一般的に、合成及び／又は貯蔵の間の、キラル制御オリゴヌクレオチドの安定性を向上させる。サイクルの後、脱保護／放出の間、ホスホロチオエートジエステル前駆体はホスホロチオエートジエステル結合に変換される。いくつかの実施形態では、キラル制御オリゴヌクレオチドにリン酸ジエステル結合が存在しないとき、又は、合成の間酸化ステップが必要でないときでも、ホスホロチオエートジエステル前駆体を用いることは有益である。 Both phosphorothioates and phosphate diester bonds can be incorporated into the same chiral controlled oligonucleotide as exemplified in Scheme Id. As will be appreciated by those skilled in the art, the methods provided do not require a contiguous phosphodiester and phosphate diester, and other internucleotide bonds between the two are formed using the cycle described above. Can be done. In Scheme Id, the phosphodiester precursor precursor,

Is introduced in place of the phosphodiester bond. In some embodiments, such replacement provided an improvement in synthetic efficiency during a given step, eg, an oxidation step. In some embodiments, the use of phosphodiester precursor precursors generally improves the stability of chiral controlled oligonucleotides during synthesis and / or storage. After the cycle, during deprotection / release, the phosphodiester precursor is converted to a phosphodiester bond. In some embodiments, it is beneficial to use phosphodiester precursors even in the absence of phosphate diester bonds in chiral controlled oligonucleotides or when oxidation steps are not required during synthesis.

の構造を有する。 As in Scheme Ic, in some embodiments, non-chiral pure phosphoramidite can be used in cycles involving oxidation steps. In some embodiments, DMTr protected β-cyanoethyl phosphoramideite is used. In some embodiments, the phosphoramidite used is

Has the structure of.

In some embodiments, the methods of the invention provide chiral controlled oligonucleotide compositions that are enriched to a particular oligonucleotide type.

In some embodiments, at least about 10% of the crude composition provided is of a particular oligonucleotide type. In some embodiments, at least about 20% of the crude composition provided is of a particular oligonucleotide type. In some embodiments, at least about 30% of the crude composition provided is of a particular oligonucleotide type. In some embodiments, at least about 40% of the crude composition provided is of a particular oligonucleotide type. In some embodiments, at least about 50% of the crude composition provided is of a particular oligonucleotide type. In some embodiments, at least about 60% of the crude composition provided is of a particular oligonucleotide type. In some embodiments, at least about 70% of the crude composition provided is of a particular oligonucleotide type. In some embodiments, at least about 80% of the crude composition provided is of a particular oligonucleotide type. In some embodiments, at least about 90% of the crude composition provided is of a particular oligonucleotide type. In some embodiments, at least about 95% of the crude composition provided is of a particular oligonucleotide type.

In some embodiments, at least about 1% of the provided composition is a particular oligonucleotide type. In some embodiments, at least about 2% of the provided composition is a particular oligonucleotide type. In some embodiments, at least about 3% of the provided composition is a particular oligonucleotide type. In some embodiments, at least about 4% of the provided composition is a particular oligonucleotide type. In some embodiments, at least about 5% of the provided composition is a particular oligonucleotide type. In some embodiments, at least about 10% of the provided compositions are of a particular oligonucleotide type. In some embodiments, at least about 20% of the provided composition is a particular oligonucleotide type. In some embodiments, at least about 30% of the provided composition is a particular oligonucleotide type. In some embodiments, at least about 40% of the provided compositions are of a particular oligonucleotide type. In some embodiments, at least about 50% of the provided compositions are of a particular oligonucleotide type. In some embodiments, at least about 60% of the provided compositions are of a particular oligonucleotide type. In some embodiments, at least about 70% of the provided compositions are of a particular oligonucleotide type. In some embodiments, at least about 80% of the provided compositions are of a particular oligonucleotide type. In some embodiments, at least about 90% of the provided compositions are of a particular oligonucleotide type. In some embodiments, at least about 95% of the provided compositions are of a particular oligonucleotide type.

Biological Applications As discussed in detail herein, the invention provides, among other things, a chiral-controlled oligonucleotide composition, which comprises a plurality of oligonucleotides of at least one type. Means to contain. Each oligonucleotide molecule of a particular "type" is in terms of (1) base sequence, (2) skeletal binding pattern, (3) skeletal chiral center pattern, and (4) skeletal P-modifying component pattern. It consists of preselected (eg, predetermined) structural elements. In some embodiments, the oligonucleotide compositions provided contain oligonucleotides prepared in a single synthetic process. In some embodiments, the provided composition is an oligo having more than one chiral composition within a single oligonucleotide molecule (eg, multiple different residues along the oligonucleotide have different steric chemistry). Containing nucleotides; in some embodiments, such oligonucleotides are in a single synthetic process without the need to generate individual oligonucleotide molecules with more than one chiral bond in a secondary binding step. Obtainable.

The oligonucleotide compositions provided herein can be used as agents to regulate some cell-related processes and mechanisms including, but not limited to, transcription, translation, immune response, epigenetics and the like. .. In addition, the oligonucleotide compositions provided herein can be used as reagents for research and / or diagnostic purposes. Those skilled in the art will readily recognize that the disclosure of the invention herein is not limited to specific uses, but can be applied to any situation in which the use of synthetic oligonucleotides is desirable. In addition to others, among other things, the provided compositions are useful in a variety of therapeutic, diagnostic, agricultural, and / or research applications.

In some embodiments, the oligonucleotide composition provided comprises an oligonucleotide and / or a residue thereof comprising one or more structural modifications described in detail herein. In some embodiments, the oligonucleotide composition provided comprises an oligonucleotide containing one or more nucleic acid analogs. In some embodiments, the provided oligonucleotide compositions are peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), glycon nucleic acid (GNA), threose nucleic acid (TNA), xenonucleic acid. (ZNA), and combinations thereof, etc., including, but not limited to, oligonucleotides containing one or more artificial nucleic acids or residues.

In any of the embodiments, the present invention is useful for oligonucleotide-based regulation of gene expression, immune response, and the like. Accordingly, the sterically defined oligonucleotide compositions of the present invention contain certain types of oligonucleotides (ie, chiral controlled and optionally chiral pure) and are conventional sterically. Can be used in place of random or chirally impure equivalents. In some embodiments, the provided composition exhibits enhanced intended effects and / or reduced unwanted side effects. Certain embodiments of the present invention for biological and clinical / therapeutic uses are explicitly discussed below.

Various dosing regimens can be utilized to administer the provided chiral controlled oligonucleotide compositions. In some embodiments, multiple unit doses are administered at time intervals. In some embodiments, a given composition has a recommended dosing regimen, which involves one or more doses. In some embodiments, the dosing regimen comprises multiple doses, each separated from each other by the same length of time; in some embodiments, the dosing regimen comprises multiple doses and at least separate doses. Includes two different times. In some embodiments, all doses within the dosing regimen have the same unit dose. In some embodiments, multiple different doses within the dosing regimen will vary in dose. In some embodiments, the dosing regimen comprises a first dose of a first dose followed by a single or multiple dose of a second dose different from the first dose. In some embodiments, the dosing regimen is a second (or subsequent) dose equal to or different from the first dose of the first dose followed by the first dose (or another previous dose). ) Includes one or more additional doses of the dose. In some embodiments, the dosing regimen comprises administering at least one unit dose per day at least. In some embodiments, the dosing regimen comprises administering more than one dose over a period of at least one day, sometimes longer than one day. In some embodiments, the dosing regimen comprises administering multiple doses over a period of at least a week. In some embodiments, the duration is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. , 22, 2324, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 weeks or more (eg, about 45, 50, 55). , 60, 65, 70, 75, 80, 85, 90, 95, 100 weeks or more). In some embodiments, the dosing regimen comprises administering a single dose per week for longer than a week. In some embodiments, the dosing regimen is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. , 22, 2324, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 weeks or more (eg, about 45, 50, 55). , 60, 65, 70, 75, 80, 85, 90, 95, 100 weeks, or more), including administration of a single dose per week. In some embodiments, the dosing regimen comprises administering a single dose per 2 weeks for longer than 2 weeks. In some embodiments, the dosing regimen is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. , 22, 2324, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 weeks or more (eg, about 45, 50, 55). , 60, 65, 70, 75, 80, 85, 90, 95, 100 weeks, or more), including administration of a single dose per 2 weeks. In some embodiments, the dosing regimen comprises administering one dose per month per month. In some embodiments, the dosing regimen comprises administering a single dose per month for longer than one month. In some embodiments, the dosing regimen is to administer a single dose per month for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or longer. Including that. In some embodiments, the dosing regimen comprises administering a once-weekly dose about every 10 weeks. In some embodiments, the dosing regimen comprises administering a single dose per week approximately every 20 weeks. In some embodiments, the dosing regimen comprises administering a dose once a week for about 30 weeks. In some embodiments, the dosing regimen comprises administering a weekly dose every 26 weeks. In some embodiments, the chiral-controlled oligonucleotide compositions are chiral-controlled (eg, sterically random) oligonucleotide compositions of the same sequence and / or different chiral-controlled oligonucleotide compositions of the same sequence. Administer according to a dosing regimen different from the dosing regimen used for. In some embodiments, the chiral-controlled oligonucleotide composition is administered according to a reduced dosing regimen compared to the dosing regimen of the non-chiral-controlled (eg, sterically random) oligonucleotide composition of the same sequence. This is because the latter achieves a lower total exposure level over a given unit time, contains one or more lower unit doses, and / or takes a smaller number of doses over a given unit time. It means to include. In some embodiments, the chiral-controlled oligonucleotide composition is administered according to a dosing regimen over a longer period than the dosing regimen of the non-chirally controlled (eg, sterically random) oligonucleotide composition of the same sequence. Although not desired to be limited by theory, in some embodiments, shorter dosing regimens and / or longer dosing durations provide stability, bioavailability of chiral controlled oligonucleotide compositions. The applicant notes that it may be due to an increase in rate and / or effectiveness. In some embodiments, the chiral-controlled oligonucleotide composition has a longer dosing regimen than an equivalent non-chirally controlled oligonucleotide composition. In some embodiments, the chiral-controlled oligonucleotide composition has a shorter duration between the two doses than an equivalent non-chirally controlled oligonucleotide composition. Although not desired to be limited by theory, in some embodiments the longer-term dosing regimen and / or shorter dosing duration is due to improved safety of the chiral-controlled oligonucleotide composition. The applicant notes that it is possible.

A single dose may contain a variety of amounts of one type of chiral controlled oligonucleotide that are appropriately desired depending on the application. In some embodiments, a single dose is about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170. , 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, or more (eg, about 350, 400, 450, 500, 550, 600, 650, 700, Contains 750, 800, 850, 900, 950, 1000, or more) mg of one type of chiral controlled oligonucleotide. In some embodiments, a single dose contains about 1 mg of one type of chiral controlled oligonucleotide. In some embodiments, a single dose contains about 5 mg of one type of chiral controlled oligonucleotide. In some embodiments, a single dose contains about 10 mg of one type of chiral controlled oligonucleotide. In some embodiments, a single dose contains about 15 mg of one type of chiral controlled oligonucleotide. In some embodiments, a single dose contains about 20 mg of one type of chiral controlled oligonucleotide. In some embodiments, a single dose contains about 50 mg of one type of chiral controlled oligonucleotide. In some embodiments, a single dose contains about 100 mg of one type of chiral controlled oligonucleotide. In some embodiments, a single dose contains about 150 mg of one type of chiral controlled oligonucleotide. In some embodiments, a single dose contains about 200 mg of one type of chiral controlled oligonucleotide. In some embodiments, a single dose contains about 250 mg of one type of chiral controlled oligonucleotide. In some embodiments, a single dose contains about 300 mg of one type of chiral controlled oligonucleotide. In some embodiments, the chiral-controlled oligonucleotide is administered in a smaller single dose and / or total dose than the non-chiral controlled oligonucleotide. In some embodiments, the chiral-controlled oligonucleotide is administered in a smaller single dose and / or total dose than the non-chiral controlled oligonucleotide, which is due to improved efficacy. In some embodiments, the chiral-controlled oligonucleotide is administered in a higher single dose and / or total dose than the non-chiral controlled oligonucleotide. In some embodiments, the chiral-controlled oligonucleotide is administered in a higher single dose and / or total dose than the non-chiral controlled oligonucleotide, which is due to improved safety.

Bioactive oligonucleotides The oligonucleotide compositions provided herein as used herein may include single-stranded and / or multi-stranded oligonucleotides. In some embodiments, the single-stranded oligonucleotide is self-hybridizing under relevant conditions so that even the single-stranded oligonucleotide used can have at least partially double-stranded properties. Includes complementary parts. In some embodiments, the oligonucleotides contained in the provided composition are single-stranded, double-stranded, or triple-stranded. In some embodiments, the oligonucleotides contained in the provided composition include single-stranded and multi-stranded moieties within the oligonucleotide. In some embodiments, as described above, each single-stranded oligonucleotide can have a double-stranded region and a multi-stranded region.

In some embodiments, the provided composition comprises one or more oligonucleotides that are completely or partially complementary to the following strands: structural genes, gene regulation and / or termination regions, and /. Or a self-replicating system, such as viral or plasmid DNA. In some embodiments, the provided composition is siRNA or other RNA interfering reagent (RNAi or iRNA agent), shRNA, antisense oligonucleotide, self-cleaving RNA, ribozyme, fragment and / or variant thereof. (For example, peptidyl transferase 23S rRNA, RNase P, Group I and Group II introns, GIR1 branched ribozyme, redzyme, hairpin ribozyme, hammerhead ribozyme, HDV ribozyme, mammalian CPEB3 ribozyme, VS ribozyme, glmS ribozyme, CoTC ribozyme, etc. ), MicroRNA, MicroRNA mimic, Super mir, Aptamer, Antimir, Antagonist, Antago mir, Ul adapter, Triple chain forming oligonucleotide, RNA activator, Long non-coding RNA, Short non-coding (eg piRNAs), With immunomodulatory oligonucleotides (immunostimulatory oligonucleotides, immunoinhibitory oligonucleotides), GNA, LNA, ENA, PNA, TNA, morphono, G-quadruplex (RNA and DNA), antiviral oligonucleotides, and decoy oligonucleotides. Includes one or more oligonucleotides that are, or act as such.

In some embodiments, the provided composition comprises one or more hybrid (eg, chimeric) oligonucleotides. In the context of the present disclosure, the term "hybrid" broadly means a mixed component of an oligonucleotide, wherein the hybrid oligonucleotide is, for example, (1) an oligonucleotide molecule having a mixed type of nucleotide, eg, a single molecule. Partial DNA and partial RNA within (eg, DNA-RNA); (2) Different types of nucleic acids such that DNA-RNA base pairing occurs intramolecularly, intermolecularly, or both. Complementary pair; (3) an oligonucleotide having two or more types of skeletal or interbase bonds.

In some embodiments, the provided composition comprises one or more oligonucleotides containing more than one nucleic acid residue in a single molecule. For example, in any of the embodiments described herein, oligonucleotides may comprise a DNA portion and an RNA portion. In some embodiments, oligonucleotides may include invariant and altered moieties.

The oligonucleotide composition provided can include, for example, an oligonucleotide containing any of the numerous modifications described herein. In some embodiments, certain changes are selected, eg, in terms of intended use. In some embodiments, it is preferred to modify one or both strands of the double-stranded oligonucleotide (or the double-stranded portion of the single-stranded oligonucleotide). In some embodiments, the double strand (or portion) comprises different modifications. In some embodiments, the duplex comprises the same modification. Those skilled in the art will appreciate that the extent and type of changes made possible by the methods of the invention can create a large sequence of changes. Examples of such changes are described herein and are not limited.

RNA Interference The oligonucleotide compositions provided are useful, among others, for application in RNA interference.

RNA interference (RNAi) means inhibition of gene expression by RNA molecules. Typically, there are small, double-stranded RNA molecules. Since gene expression regulates most cellular processes, the ability to inhibit gene expression is a potentially powerful tool for regulating biological conditions, including diseases in humans and / or animals (eg, domestic animals or pets). I will provide a. Numerous studies have been conducted to demonstrate the use of RNAi in regulating or regulating disease-related gene expression. See, for example: Cullen, KA, Hall, MJ & Golosinskiy, A. US Outpatient Surgery http://www.rxipharma.com/wp-content/uploads/2012/05/Cullen-2009.pdf, 2006. Natl Health Stat Report 2009; 1-25; Elbashir S, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T., 21 Nucleotide RNA double strands mediate RNA interference in cultured mammalian cells, Nature 2001; 411 : 494-498; Fire A, Xu S, Montgomery MK, Kostas SA, Driveri SE & Mello C. Strong and specific RNA interference by double-stranded RNA in Caenolabditis elegance, http://www.ncbi. nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9486653 Nature 1998; 391 (6669): 806-811; Gauglitz, GG, Kortin, HC, Pavicic, T., Ruzicka, T. , & Jeschke, MG, Thick Scars and / or Keroids: Pathological Mechanisms and Modern Treatments, Mol Med 2011; 17 (1-2): 113-125; Li-Tsang, CW, Lau, JC & Chan, CC , Affected by hypertrophic scar formation and its characteristics in Chinese, Burns 2005; 31, 610-616; Wang H, Ghosh A, Baigude H, Yang C, Qui L, Xia L et al., Chemically modified siRNA for mutant SOD1 Therapeutic gene silence delivered by ALS delays ALS progression, JBC 2008; 283 (23): 15845-15852; Weiser, TG, Regenbogen, SE, Thompson, KD, Haynes, AB, Lipsitz, SR, Berry, WR & Gawande, A.A., Global Surgical Estimates: Model Strategies Based on Available Data, Lancet 2008; 372 (9633): 139-44.

The phenomenon of RNA interference was first shown in C. elegans, where insertion of dsRNA molecules inhibited complementary gene expression. Although the use of siRNA has become a widely used tool for down-regulating gene expression, the existence of natural pathways in eukaryotes is well documented. The origin of the endogenous siRNA (or miRNA) may be a transposon, virus, repetitive sequence and gene. The steps to produce efficient endogenous siRNA are controlled by three enzymes. RNA-dependent RNA polymerase converts single-stranded RNA into double-stranded RNA. Alternatively, DNA-dependent RNA polymerases produce dsRNA by transcribing contralateral DNA repeat sequences. The resulting large RNA molecule is subjected to digestion with Ribonuclease III (Dicer) to produce a short double-stranded siRNA molecule. The argonaute protein is then used to form a complex known as RISC (RNA-induced silence complex). Must be bound to the siRNA molecule. RISC recognizes a double-stranded RNA fragment, divides the double strand into two, and maintains the single strand in the RISC complex. RISC can then promote epigenetic silencing by RNA-oriented DNA methylation or target RNA cleavage. Protein translation can be significantly knocked down, but siRNAs usually do not completely rule out expression of gene targets. Thus, RISC can bind and disrupt the RNA guide strand to its corresponding cellular messenger RNA target. Thus, RNAi provides a method of potentially blocking the production of disease-causing proteins.

siRNA technology presents useful molecular tools. The use of RNA interference to artificially manipulate gene expression was originally limited by the activation of cellular antiviral mechanisms. It has been shown that exposure of cells to sequences over 30 nucleotides in length induces interferon gene expression, leading to non-specific RNA degradation and reduced protein synthesis. However, this problem can be circumvented by short (eg, 19-22 nucleotides) siRNA sequences. Methods for delivering siRNA to cells include, without limitation, liposomal addition of purified ribonucleotides to the vehicle, or transfection of a plasmid vector designed to express the siRNA molecule. The plasmid vector relies on the use of two RNA polymerase III promoters (U6 and H1) to drive the transcription of siRNA molecules. The target sequence (19-29 nucleotides) is placed in the sense and antisense directions with a group of small spacers between (short hairpin RNA or shRNA). Once transcribed, a hairpin structure that can be recognized and cleaved by Dicer is formed. Alternatively, the RNA duplex is transcribed without a hairpin structure and processed directly by RISC. Currently, there are various plasmids and viral vectors that utilize similar concepts to generate siRNA, shRNA, or single-stranded siRNA (ss-siRNA) molecules (see, eg, 2012 Cell-150-883). Walt Lima et al., SsRNAi activates RNAi in animals).

In some embodiments, the oligonucleotide or oligonucleotide composition provided is useful as an ss-siRNA or GalNAc complex siRNA.

The technique is familiar with certain structural features that affect siRNA as a tool. After the discovery of siRNA, numerous studies were attempted to identify the optimal features required for siRNA design. Requirements include the use of sequences shorter than 30 nucleotides to avoid PKR activation, sequence stability at the 5'end of the antisense strand to the 3'end, and insertion of a TT overhang. Based on studies such as these, a number of algorithms have been developed by academic and industrial laboratories to predict the most efficient target sequences for a given gene. Most of these programs are not perfect, but the chances of getting the predicted sequence are higher than designing the sequence without considering the recommended features. Multiple sequence synthesis and testing may be required. The design of siRNA experiments involves some potential pitfalls and should be designed to include suitable control and measurable endpoints. Negative regulation can include non-complementary sequences that have thermodynamically similar properties to valid siRNA sequences. When introducing siRNA or shRNA using a plasmid vector as a transfection, the ratio of lipid to nucleic acid may be equal, and the control vector may contain a sequence that is transcribed and processed intracellularly. Assessment of SiRNA effects can also be performed by measuring both RNA and protein expression.

In certain embodiments, the oligonucleotides provided herein are double-stranded. Double-stranded oligonucleotides, typically containing 20-23 double-stranded structures, specifically 21 base pairs, have been evaluated to be particularly effective in inducing RNA interference (Elbashir). Et al., EMBO 2001, 20: 6877-6888). However, shorter or longer double-stranded oligonucleotides have also been found to be effective.

In some embodiments, the double-stranded oligonucleotide used in accordance with the present invention comprises two oligonucleotide chains that are sufficiently complementary to be able to hybridize to form a double-stranded structure. In some embodiments, the duplex is about 12 to about 45 base pair length. In some embodiments, the duplex is about 18 to about 25 base pair length. In some embodiments, the duplex is about 19 to about 24 base pair length. In some embodiments, the duplex is about 19 to about 21 base pair length. In some embodiments, the duplex is a duplex oligonucleotide of about 25 to about 30 base pairs in length. In some embodiments, the duplex is a duplex oligonucleotide of about 10 to about 15 base pairs in length. In some embodiments, the duplex is at least about 21 bases long.

In some embodiments, the double-stranded oligonucleotide utilized in accordance with the present invention comprises a sense strand and an antisense strand, the region of complementarity in which the antisense RNA strand is complementary to at least a portion of the target sequence. The double-stranded region is about 14 to about 30 nucleotides in length. In some embodiments, the complementarity regions to the target sequence are about 14 to about 30 nucleotides in length. In some embodiments, the complementarity regions to the target sequence are about 18 to about 25 nucleotides in length. In some embodiments, the complementarity regions to the target sequence are about 19 to about 24 nucleotides in length. In some embodiments, the complementarity regions to the target sequence are about 19 to about 21 nucleotides in length.

As used herein, the term "antisense strand" means an oligonucleotide that is substantially or 100% complementary to the target sequence of interest. The term "antisense strand" includes the antisense region of both oligonucleotides formed from two separate strands, and a single molecule oligonucleotide capable of forming a hairpin or dumbbell-shaped structure. The terms "antisense" and "guide strand" are used interchangeably herein.

The term "sense strand" means an oligonucleotide that, in whole or in part, has the same nucleotide sequence as the target sequence, such as the sequence of messenger RNA or DNA. The terms "sense strand" and "passenger strand" are used interchangeably herein.

"Target sequence" means any nucleic acid sequence whose expression or activity is regulated. The target nucleic acid may be DNA or RNA, such as endogenous DNA or RNA, viral DNA or viral RNA, or other RNA, virus, bacterium, fungus, mammal, or plant encoded by the gene. In some embodiments, the target sequence is associated with a disease or disorder.

"Can be specifically vibrated" and "complementary" mean that a nucleic acid forms hydrogen bonds with another nucleic acid, either by a typical Watson click or other atypical form. Means that you can. In connection with the nucleic acid molecule of the present invention, the binding free energy of the nucleic acid molecule and its complementary sequence is sufficient for promoting the relevant function of the nucleic acid, eg RNAi activity. Determination of the binding free energy of nucleic acid molecules is well known in the art (eg Turner et al., 1987, CSH Symp. Quant. Biol. LIT pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA83: 9373-9377; Turner et al., 1987, /. See Ain. Chem. Soc. 109: 3783-3785).

Percentage of complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form a hydrogen bond (eg, Watson-Crick base pair) with a second nucleic acid sequence (eg, 5, 6, 7, out of 10). 8, 9, 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Complete complementarity" or 100% complementarity means that all contiguous residues of a nucleic acid sequence will form hydrogen bonds with contiguous residues in the same number of second nucleic acid sequences. Incomplete complementarity means a state in which some, but not all, of the nucleoside units of the two chains can form hydrogen bonds with each other. By "substantially complementary" is meant a polynucleotide chain exhibiting 90% or more complementarity, except for polynucleotide chain regions such as overhangs, which are selected to be non-complementar. Specific binding to a non-target sequence of an oligomer compound under physiological conditions where specific binding is desired, for example in the case of an in vivo assay or therapeutic method, or in the case of an in vitro assay under conditions in which the assay is performed. A sufficient degree of complementarity is required to avoid non-specific binding. In some embodiments, the non-target sequence differs from the corresponding target sequence by at least 5 nucleotides.

Double-chain oligonucleotides In some embodiments, double-chain oligonucleotides used in accordance with the present invention are used by endogenous molecules such as Dicer to produce smaller double-chain oligonucleotides, such as RNAi agents. Large enough to be cleaved. In some embodiments, the provided double-stranded oligonucleotide regulates the expression of the target gene by RISC-mediated cleavage of the target sequence.

In some embodiments, the double chain region of the double chain oligonucleotide is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotide pairs in length or equivalent.

In some embodiments, the antisense strand of the double-stranded oligonucleotide is at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28. , 29 or 30 nucleotides in length, or equal.

In some embodiments, the sense strand of the double-stranded oligonucleotide is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length or equivalent.

In some embodiments, one strand has at least one stretch of 1-5 single strand nucleotides in the double chain region. "Stretching of single-stranded nucleotides in a double-stranded region" means the presence of at least one nucleotide base pair at both ends of the single-stranded stretch. In some embodiments, the two strands have at least one stretch of 1-5 (eg, 1, 2, 3, 4 or 5) single-stranded nucleotides in the double chain region. When two strands have stretches of 1-5 single-stranded nucleotides in the double-stranded region, can such single-stranded nucleotides be opposite to each other (eg, mismatched stretch), or first. The two strands may be positioned to have no single strand nucleotide opposite to the first strand single strand oligonucleotide and vice versa (eg, single strand loop). In some embodiments, the single-stranded oligonucleotide is within 8 nucleotides from either end, eg, 8, 7, 6, from any of the 5'or 3'ends of the complementarity regions between the two strands. It is present within 5, 4, 3 or 2 nucleotides.

In some embodiments, each strand of the double-stranded oligonucleotide used in accordance with the present invention is an international patent application filed on March 8, 2004, the entire contents of which are incorporated herein. It has a ZXY structure as described in PCT / US 2004/07070.

Hairpins and Dumbbells In some embodiments, the double chain oligonucleotide used according to the invention is a single molecule containing self-complementarity regions; thus the two "chains" of the double chain regions are in fact. Are covalently bonded to each other. Such two chains may bind to each other at both ends or only one end. Binding at one end means that the 5'end of the first chain is attached to the 3'end of the second chain, or the 3'end of the first chain is attached to the 5'end of the second chain. means. When the two chains are attached to each other at both ends, the 5'end of the first chain is attached to the 3'end of the second chain and the 3'end of the first chain is attached to the 5'end of the second chain. Will be done. In some embodiments, the two strands are (N) _n ; N together by an oligonucleotide linker, including but not limited to those in which N is an independently modified or unmodified nucleotide and n is 3-23. Combined with. In some embodiments, n is 3-10, such as 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the oligonucleotide linkers are GNRA, (G) ₄ , (U) ₄ and (dT) ₄ (where N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide). Is selected from the group consisting of). In some embodiments, some of the nucleotides in the linker are involved in base pair interactions with other nucleotides in the linker. In some embodiments, the two chains are attached to each other by a non-nucleoside linker.

In some embodiments, hairpin and dumbbell RNAi agents have at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24 or 25 nucleotide pairs or equivalent double chains. Have. In some embodiments, the double chain region is 200, 100 or 50 nucleotides pair length or less. In some embodiments, the range of the double chain region is about 15 to about 30, about 17 to about 23, about 19 to about 23, and about 19 to about 21 nucleotide pairs in length. In some embodiments, hairpin oligonucleotides mimic the natural precursors of microRNAs.

In some embodiments, the hairpin RNAi agent may have, for example, a single-stranded overhang or end-unpaired region at the 3'end on the antisense side of the hairpin. In some embodiments, the overhang is about 1 to about 4 nucleotides in length. In some embodiments, the overhang is about 2-3 nucleotides in length.

In some embodiments, the hairpin RNAi agent is characterized in that the 3'end of the antisense strand is attached to the 5'end of the sense strand. In some embodiments, the hairpin RNAi agent is characterized in that the 5'end of the antisense strand is attached to the 3'end of the sense strand. The hairpin oligonucleotides provided are also referred to herein as "shRNA".

Single-stranded oligonucleotides In some embodiments, single-stranded oligonucleotides used in accordance with the present invention are substantially complementary to the "sense" nucleic acid encoding the gene expression product, eg, coding a double-stranded cDNA molecule. Includes nucleic acid sequences that are complementary to the strands or that are complementary to RNA sequences such as pre-mRNA, mRNA, miRNA or pre-miRNA. The single-stranded oligonucleotides provided include, but are not limited to, antisense oligonucleotides and single-stranded RNAi agents. In some embodiments, the complementarity regions have a nucleotide length of less than about 30. In some embodiments, the complementarity regions have a nucleotide length of at least less than about 15. In some embodiments, the provided single-stranded oligonucleotides are about 10 to about 25 nucleotides in length (eg, about 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length). In some embodiments, the provided single-stranded oligonucleotide is about 25 to about 30 nucleotides in length. In some embodiments, the provided single-stranded oligonucleotide is about 15-about 29 nucleotides in length. In some embodiments, the provided single-stranded oligonucleotide is characterized by having less than 100% complementarity to mRNA, RNA or DNA arc. In some embodiments, the provided single-stranded oligonucleotide has a ZXY structure as described in International Patent Application No. PCT / US 2004/07070 filed March 8, 2004.

In some embodiments, the single-stranded oligonucleotide used can hybridize to a complementary RNA, such as mRNA, pre-mRNA, preventing access of the translation mechanism to the target RNA transcript, thereby. It can block protein synthesis. In some embodiments, the provided single-stranded oligonucleotide can hybridize to complementary RNA and the RNA target can result in cleavage by an enzyme such as RNasc H, thus translating the target RNA. Stop. In some embodiments, the provided single-stranded oligonucleotide regulates the expression of the target gene by RISC-mediated cleavage of the target sequence.

As used herein, a "single-stranded RNAi agent" is an RNAi agent consisting of a single molecule. In some embodiments, the single-stranded RNAi agent comprises or comprises a double-stranded region formed by intra-strand base pairing, eg, a hairpin or frying pan stalk structure. In some embodiments, the single-stranded RNAi agent is antisense with respect to the target molecule. In some embodiments, the single-stranded RNAi agent is long enough to enter RISC and participate in RISC-mediated cleavage of the target mRNA. An exemplary single-stranded siRNA (ss siRNA) is known and is described, for example, in US Patent Application Publication No. 2006/0166901, which is incorporated herein by reference in its entirety.

In some embodiments, the single-stranded RNAi agent is at least about 12 nucleotides in length. In some embodiments, the single-stranded RNAi agent is at least about 15 nucleotides in length. In some embodiments, the single-stranded RNAi agent is at least about 20 nucleotides in length. In some embodiments, the single-stranded RNAi agent is at least about 25 nucleotides in length. In some embodiments, the single-stranded RNAi agent is at least about 29 nucleotides in length. In some embodiments, the single-stranded RNAi agent is at least about 30 nucleotides in length. In some embodiments, the single-stranded RNAi agent is at least about 35 nucleotides in length. In some embodiments, the single-stranded RNAi agent is at least about 40 nucleotides in length. In some embodiments, the single-stranded RNAi agent is at least about 50 nucleotides in length.

In some embodiments, the single-stranded RNAi agent has a nucleotide length of less than 200. In some embodiments, the single-stranded RNAi agent has a nucleotide length of less than 100. In some embodiments, the single-stranded RNAi agent has a nucleotide length of less than 60.

In some embodiments, the single-stranded RNAi agent is 5'phosphorylated. In some embodiments, the single-stranded RNAi agent comprises a phosphoryl analog at the 5'prime end. In some embodiments, the single-stranded RNAi agent has a length of about 15 to about 29 nucleotides in length.

Single-stranded oligonucleotides containing single-stranded siRNAs, microRNAs or mir and / or identified oligonucleotides that can be used as targets or templates for the design of the oligonucleotides of the invention. Esau et al., Publication No. 20050261218, entitled "oligonucleotides and compositions for use in the regulation of small non-coding RNA," which is incorporated herein by reference in its entirety. It is taught in USSN: 10/909125).

The present invention includes research and / or diagnostic reagents comprising single-stranded oligonucleotides. In some embodiments, the single-stranded oligonucleotide used in accordance with the present invention is and / or acts as a primer. In some embodiments, the primer is used in a polymerase chain reaction (ie, PCR) to amplify the nucleic acid. These applications include any known variants of PCR such as reverse transcription PCR (RT-PCR) and real-time PCR.

MicroRNA
In some embodiments, the provided composition comprises one or more oligonucleotides that are or act as microRNAs.

MicroRNAs (miRNAs or mirs) are highly conserved types of small RNA molecules transcribed from DNA in the genomes of plants and animals, but are not translated into proteins. Pre-microRNAs are processed into miRNAs. Processed microRNAs are integrated into RNA-induced silencing complex (RISC) and are identified as key regulators of development, cell proliferation, apoptosis and differentiation, single-stranded 17-25 nucleotides (1 strand). nt) RNA molecule. They are thought to play an important role in the regulation of gene expression by binding to the 3'untranslated regions of specific mRNAs. RISC mediates downregulation of gene expression by translational inhibition, transcriptional cleavage, or both. RISC is also involved in transcriptional silencing in the nucleus of a wide range of eukaryotes.

MicroRNAs have also been associated with the regulation of pathogens in the host. See, for example, Jopling, C.L. et al., Science (2005) vol. 309, pp 1577-1581. Without being bound by theory, administration of microRNAs, microRNA mimics, and / or anti-microRNA oligonucleotides results in regulation of pathogenic survival, growth, development, and / or replication. In some embodiments, the provided oligonucleotides are microRNAs, microRNA mimics, and / or anti-microRNAs, where the microRNAs are host microRNAs. The number of miRNA sequences identified so far is numerous and increasing, for example, "miRBase: Micro RIVA Sequences, Targets and Gene Technicians" Griffiths-Jones S, Grocock RJ, van. Dongen S, Bateman A, Enright AJ. NAR, 2006, 34, Database Issue, D140-D144; Found in "The microRNA Registry" Griffiths-Jones S. NAR, 2004, 32, Database Issue, D109-D111. Non-limiting examples of useful miRNAs are also provided in Attachment (C).

Ribozyme In some embodiments, the composition provided comprises one or more oligonucleotides that are or act as ribozymes.

Ribozymes are oligonucleotides with specific catalytic domains with endonuclease activity (Kim and Cech, Proc Natl Acad Sci US A. 1987 Dec; 84 (24): 8788-92; Forster and Symons, Cell. 1987. Apr 24; 49 (2): 211-20). At least 6 basic variants of the natural enzyme RNA are currently known. In general, enzymatic nucleic acids function by first binding to the target RNA. Such binding occurs via the target binding portion of the enzymatic nucleic acid, which is maintained in the immediate vicinity of the enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid acts to recognize the target RNA first, then bind by complementary base pairing, and once bind to the modification site, enzymatically cleave the target RNA. Strategic cleavage of such target RNA would disrupt its ability to synthesize the encoded protein directly. After the enzymatic nucleic acid binds to its RNA target and cleaves the target, it is released from the RNA to search for another target and repeatedly binds to and cleaves the new target.

Methods of producing ribozymes that target arbitrary target sequences are known in the art. Ribozymes can be specifically designed as described in WO 93/23569 and WO 94/02595, each of which is specifically incorporated herein by reference. Synthesized to be tested in vitro and in vivo as described herein).

Aptamers In some embodiments, the provided composition comprises one or more oligonucleotides that are or act as aptamers.

Aptamers are nucleic acid or peptide molecules that bind to specific molecules of interest with high affinity and specificity (Tuerk and Gold, Science 249: 505 (1990); Ellington and Szostak, Nature 346: 818 (1990)). .. DNA or RNA aptamers that bind to many different substances, from large proteins to small organic molecules, have been successfully produced. Eaton, Curr. Opin. Chem. Biol. 1: 10-16 (1997), Famulok, Curr. Opin. Struct. Biol. 9: 324-9 (1999), and Hermann and Patel, Science 287: 820-5 ( 2000) See. The aptamer may be RNA type or DNA type. In general, aptamers are selected in vitro or equivalent iterations, ie, SELEX (in vitro man-made) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Produced by evolutionary method). The SELEX method is a protocol in which single-stranded oligonucleotides are selected from a vast sequence library based on their binding affinity for the target protein or other molecule (C. Tuerk, L. Gold, Science, 249). (1990), pp. 505-510; R. Green, AD Ellington, DP Bartel, JW Szostak, Methods Enzymol., 2 (1991), pp. 75-86; L. Gold, B. Polisky, O. Uhlenbeck, M. Yarus, Annu. Rev. Biochem., 64 (1995), pp. 763-797). The SELEX method is usually initiated with an RNA or DNA library consisting of random oligonucleotide sequences of numbers 10 ¹⁴ to 10 ¹⁵ . In a well-randomized oligonucleotide library, each molecule will exhibit unique tertiary structure that will depend on the nucleotide sequence of the molecule. The binding affinity of the oligonucleotide for the target protein will be determined by the fit between the portion on the surface of the oligonucleotide and the epitope on the target protein. As a result of starting with a vast library of diversity, it is possible to identify aptamers that have nM or sub-nM affinity for the target protein and selectivity for the target protein over other proteins with a high degree of structural homology. Usually possible (KW Uphoff, SD Bell, AD Ellington, Curr. Opin. Struct. Biol., 6 (1996), pp. 281-288). Using the SELEX method, RNA or DNA aptamers are small molecules containing multiple proteins, peptides and dopamine (C. Mannironi, A. Di Nardo, P. Fruscoloni, GP Tocchini-Valentini, Biochemistry, 36 (1997), pp. . 9726-9734), Substance P (D. Nieuwlandt, M. Wecker, Biochemistry, 34 (1995), pp. 5651-5659), Subtilisin (H. Takeno, S. Yamamoto, T. Tanaka, Y. Sakano, Y. Kikuchi, J. Biochem., 125 (1999), pp. 1115-1119), Growth Factor-Derived Thrombosis (LS Green, D. Jellinek, R. Jenison, A. Ostman, CH. Heldin, N. Janjic Biochemistry, 35 (1996), pp. 14413-14424), Vascular Endothelial Cell Growth Factor (LS Green, D. Jellinek, C. Bell, LA Bebe, BD Feistner, SC Gill, FM Jucker, N. Janjic Chem. Biol., 2 (1995), pp. 683-695), Trombin (LC Bock, LC Griffen, JA Latham, EH Vermaas, JJ Toole, Nature, 355 (1992), pp. 564-566), and L-Selectin (D. O). 'Connell, A. Koenig, S. Jennings, B. Hicke, HL Han, T. Fitzwater, YF Chang, N. Varki, D. Parma Proc. Natl. Acad. Sci. USA, 93 (1996), pp. 5883 -5887).

A number of modified SELEX protocols have been developed, as reviewed in more detail in Dua et al. (2008) Recent Patents on DNA & Gene Sequences, 2: 172-186 (incorporated herein by reference). Has been done. Non-limiting examples of the modified SELEX method are described in the following publications: Counter SELEX (US5580737), Flowcel SELEX (WO9833941), Cutting SELEX (WO0056930), Fusion SELEX (US5683867), Transctiption. ) None SELEX (US20026387620), Solution SELEX (US5567588), Chimera SELEX (WO9604403), Tissue SELEX (US20026376474), Photo SELEX (US6001577), Toggle SELEX (US20077312325), Covalent bond SELEX / Chemiselex (US5763595), Genome SELEX (US20016261774), SELEX without purified protein (WO0224954), CE-SELEX (WO03102212), Mirror SELEX-Spiegelmer (EP1386972), and Laser SELEX, DeSELEX (WO07035532), each of which is herein by reference. Incorporated as a whole). The sterically identified oligonucleotides included by the present invention can be used in any one or more variants of the SELEX method.

Aptamers can be prepared by any known method, including synthetic, recombinant and purification methods, and can be used alone or in combination with other aptamers specific for the same target. Further, as more fully described herein, the term "aptamer" specifically comprises a consensus sequence obtained by comparing two or more known aptamers to a given target. Includes "secondary aptamers". Aptamers have several characteristics that make them attractive therapeutic agents. Both DNA and RNA aptamers have been shown to bind to their targets with low to low nanomolar dissociation constants (Kd). Aptamer binding is a highly specific interaction that can even identify related proteins that share a common structural domain. Both aptamers and antibodies have similar binding affinities, but in many cases have a number of additional features that overwhelm their competitors. Unlike antibodies, aptamers can even target non-immunogenic targets. Aptamers can be readily subjected to chemical modifications during synthesis that improve their stability and pharmacokinetics. Aptamers exhibit zero or negligible levels of immunogenicity in therapeutic amounts due to their similarity to endogenous molecules.

In some embodiments, oligonucleotides used in accordance with the present invention are useful as anti-pathogenic agents, such as antiviral agents, antibacterial agents, antifungal agents and the like. Suitable targets for various pathogens such as viruses and bacteria have already been reported. For example, US Pat. Nos. 5726017, 5654151, 5469938, 5503978, 5587468, 5257894, US Patent Applications, each of which is incorporated herein by reference in its entirety. Publication No. 2005233317, US Patent No. 5496938, International Patent Application Publication No. WO08 066231, Japanese Patent Application Publication No. 2002165594, International Patent Application Publication No. WO02081494, US Patent No. 5861501, International Patent Application Publication No. WO9720072, See US Patent No. 5475096, No. 6569630, Chinese Patent Application Publication No. 101109013, and International Patent Application Publication No. WO 03106476.

In some embodiments, the oligonucleotides used in accordance with the present invention are or serve as anti-cancer agents. Aptamers include any known cancer-or tumor-related factors, such as proteins involved in the regulation of cell division or proliferation, cell cycle progression, apoptosis, cell migration, DNA repair, etc., including structural proteins and signaling molecules. Can be targeted by. Examples are, for example, Australian Patent No. 775412, US Patent No. 6232071, International Patent Application Publication No. WO 08/028534, US Pat. No. 6933114, International Patent, each of which is incorporated herein by reference in its entirety. Including Publication No. WO 04/081574, Australian Patent No. 242462, US Patent No. 6995249, No. 5668264, No. 6699843, International Patent Application Publication No. WO 04/0994614, and US Patent No. 5846713. However, it is not limited to these.

In some embodiments, the oligonucleotides used in accordance with the present invention are or serve as antiangiogenic agents. Non-limiting examples of antiangiogenic agents are VEGF and associated receptors, as well as extracellular fluid matrices or adhesion molecules. See, for example, US Pat. No. 6051698, US Patent Application Publication No. 2003175703, WO 95/21853 and US Pat. No. 7094535, each incorporated herein by reference in its entirety.

In some embodiments, the oligonucleotides used in accordance with the present invention are or serve as anticoagulants. Much is known about blood coagulation factors and their functions, as well as protein regulation such as processing. Aptamers are useful in targeting one or more factors in treating symptoms such as cardiovascular disease and impaired blood coagulation. For example, International Patent Application Publication No. WO 06/033854, US Pat. No. 5,543293, International Patent Application Publication No. WO 07/025049, US Pat. No. 6,774,118, each of which is incorporated herein by reference in its entirety. , See International Patent Application Publication No. WO 07/140000.

In some embodiments, the oligonucleotides used in accordance with the present invention are or serve as immunomodulators. Aptamers are designed to target immunomodulatory molecules associated with autoimmune diseases. The present invention may be useful for targeting molecules expressed on immune cells such as T cells and antigen presenting cells (APCs). See, for example, US Pat. No. 5,869,461, International Patent Application Publication No. WO 01/09160, each of which is incorporated herein by reference in its entirety. In some embodiments, molecules associated with the complement system such as C1, C4, C2, C3 and C5 can be targeted by aptamers. The complement system is associated with numerous renal, rheumatic, neurological, skin, blood, allergic, infectious and other diseases. See, for example, WO 97/28178 and WO 07/103549, each of which is incorporated herein by reference in its entirety. Other immune-related targets include IL-12, IL-23 and IL-28 (see, eg, WO 07/035922, International Patent Application Publication No. WO 07/035922, which is incorporated herein by reference in its entirety), IgE (eg, eg. International Patent Application Publication No. WO 05/113813, which is incorporated herein by reference in its entirety), Sp-1 and Sp1-, CD28, IL-2, GMCSF (eg, herein by reference in its entirety). US Pat. No. 6994959), SDF-1, CXCR4 (see, eg, International Patent Application Publication No. WO 08/009437, incorporated herein by reference in its entirety), IL-6, IL- 12, including, but not limited to, IFN γ (see, eg, US Pat. No. 6,599,940 and US Patent Application Publication No. 2003125279, each of which is incorporated herein by reference in its entirety), and TLR. Aptamers with identified sterics can exert an improved effect on such diseases.

In some embodiments, the oligonucleotides used in accordance with the present invention are or serve as anti-inflammatory agents. Inflammatory diseases such as acute respiratory distress syndrome (ARDS), infectious shock, emphysema, cystic fibrosis, rheumatoid arthritis and chronic bronchitis have neutrophil elastase associated with their etiology. Therefore, human elastase is a target for treating such diseases with aptamers that regulate inflammation. Other suitable targets are phosphoripase A2, such as non-pancreatic PLA2 (see WO 96/27604, International Patent Application Publication No. WO 96/27604, which is incorporated herein by reference), E-, P- and L-. Selectin (see US Pat. No. 5,780,228, which is incorporated herein by reference), cell adhesion molecules expressed in cells such as three homologous C-type lectins, leukocytes, endothelial cells, platelets and others; MCP- 1 (see International Patent Application Publication No. WO 07/093409, which is incorporated herein by reference in its entirety), NF-κB and NF-IL6 (incorporated herein by reference in its entirety, international patent application. (See Publication No. WO 00/24404), but not limited to these. Aptamers with identified sterics can exert an improved effect on such diseases.

In some embodiments, oligonucleotides used in accordance with the present invention are useful for treating specific brain diseases including, but not limited to, transmissible spongiform encephalopathy (TSE), and Alzheimer's disease. .. Known targets include International Patent Application Publication No. WO 2006/138676 and German Patent Application Publication No. 19916417, and International Patent Application Publication No. WO 08/008884, each of which is incorporated herein by reference in its entirety. It is described in the including publications. Aptamers with identified sterics can exert an improved effect on such diseases.

Decoy Oligonucleotides In some embodiments, the composition provided comprises one or more oligonucleotides that are or act as decoy oligonucleotides.

Because transcription factors recognize their relatively short binding sequences, short oligonucleotides with a consensus binding sequence for a particular transcription factor, even in the absence of surrounding genomic DNA, manipulate gene expression in living cells. Can be used as a tool for This strategy involves intracellular delivery such as "decoy oligonucleotides" that are subsequently recognized and bound by targeting factors. Occupation of the DNA binding site of a transcription factor by the decoy results in the inability of the transcription factor to bind to the promoter region of the target gene. Decoys can be used as therapeutic agents for either inhibiting the expression of genes activated by transcription factors or upregulating genes that are suppressed by transcription factor binding. Examples of the use of decoy oligonucleotides are found in Mann et al., J. Clin. Invest., 2000, 106: 1071-1075, which are expressly incorporated herein by reference in their entirety.

miRNA Mimic In some embodiments, the composition provided comprises one or more oligonucleotides that are or act as miRNA mimics.

A miRNA mimic represents a molecule that can be used to mimic a gene that regulates the activity of one or more miRNAs. Thus, the term "microRNA mimic" means a synthetic non-coding RNA that can enter the RNAi pathway and regulate gene expression (ie, miRNAs are not obtained by purification from the origin of endogenous miRNAs). miRNA mimics can be designed as mature molecules (eg, single strands) or mimetic precursors (eg, pre- or pre-miRNA).

In some embodiments, the miRNA mimic is a double-stranded molecule (eg, having a double-stranded region about 16-about 31 nucleotides in length) and has identity with the mature strand of a given miRNA. Contains more sequences.

In some embodiments, the miRNA mimic comprises a double chain region of about 16 to about 31 nucleotides. In some embodiments, the provided miRNA mimic may comprise one or more of the following chemical modification patterns: the sense strand of nucleotides 1 and 2 (counting from the 5'end of the sense oligonucleotide): Includes 2'-O-methyl modifications, as well as all of C and U; antisense strand modifications include all 2'F modifications of C and U, 5'end phosphorylation of the oligonucleotide, and 2 nucleotide 3'over. Includes stabilized internucleotide binding associated with hangs.

Super mir
In some embodiments, the provided composition comprises one or more oligonucleotides that are or act as super mir.

Supermir means an oligonucleotide, eg, a single-stranded, double-stranded or partially double-stranded oligonucleotide, having a nucleic acid sequence that is substantially identical to the miRNA and is antisense with respect to its target. .. The term includes oligonucleotides containing at least one non-natural moiety that also functions. In some embodiments, the super mir does not include the sense strand. In some embodiments, the supermir does not self-hybridize to a significant extent. In some embodiments, the supermir has a secondary structure but is substantially single chain under physiological conditions. Super mir, which is substantially single-stranded, has less than 50% (eg, about 40%, 30%, 20%, 10% or less than 5%) of super mir, which is itself double-stranded to some extent. It is a chain. The supermir may contain hairpin fragments, eg, the sequence at the 3'end is preferably self-hybridized and has a double chain region, eg, at least 1, 2, 3 or 4, preferably 8, 7, 6 or 5 nucleotides. For example, it can form a double chain of 5 nucleotides. The double chain region can be linked by a linker, such as a nucleotide linker, such as 3, 4, 5 or 6 dT, such as a modified dT. In some embodiments, the supermir is, for example, either the non-terminal or central 3'and / or 5'ends of the supermir, eg, 5, 6, 7, 8, 9 or 10 nucleotides in length. Duplexed with shorter oligonucleotides.

Antimer or miRNA Inhibitor In some embodiments, the composition provided comprises one or more oligonucleotides that are or act as antimer or miRNA inhibitor.

The terms "antimer," "microRNA inhibitor," or "miR inhibitor" are synonymous and mean oligonucleotides or modified oligonucleotides that interfere with the activity of a particular miRNA. Inhibitors can employ a variety of structures, including single-stranded, double-stranded (RNA / RNA or RNA / DNA double-stranded), and hairpin designs. In some embodiments, the microRNA inhibitor comprises one or more sequences or portions of the sequence that are wholly or partially complementary to the mature strand (or multiple mature strands) of the target miRMA. include. In some embodiments, the miRNA inhibitor comprises an additional sequence located at 5'and 3'to the sequence that is the reverse complement of mature miRMA. The additional sequence may be an inverse complement of the sequence adjacent to the mature miRNA in the premiRNA from which the mature miRNA is derived, or the additional sequence may be optional (with a mixture of A, G, C, U or dT). Can be an array of. In some embodiments, one or both of the additional sequences is any sequence that can form a hairpin. Thus, in some embodiments, the sequences that are the inverse complement of the miRNA are flanked by the hairpin structure on the 5'and 3'sides. In some embodiments, the microRNA inhibitor is double-stranded. In some embodiments, the microRNA inhibitor is double-stranded and comprises a mismatch between the nucleotides of the opposite strand. In some embodiments, the microRNA inhibitor is linked to a complex moiety to facilitate the uptake of the inhibitor into cells.

MicroRNA inhibitors, including hairpin miRNAs, are incorporated herein by reference in their entirety, Vermeulen et al., "Double-Stranded Regions Are Essential Design Components Of Potent Inhibitors of RISC Function," RNA 13: 723-730. (2007), and International Patent Application Publication Nos. W02007 / 095387 and WO 2008/036825.

Exemplary applications of miRNA-type therapies are incorporated herein by reference, Pan et al. (2007) World J Gastroenterol 13 (33): 4431-36, “New Treatments for Hepatitis C Based on Small RNAs”. It is described in "Opportunities". In short, the author describes a 22-nucleotide mature miR-122 obtained from a non-coding polyadenylated RNA transcript of the hcr gene, a liver-specific developmental regulator. Since miR-122 is a liver-specific miRNA associated with HCV viral replication, silencing of miR-122 may be useful for the treatment of HCV.

Antago mir
In some embodiments, the provided composition comprises one or more oligonucleotides that are or act as antago mir.

Antago mir is an RNA-like oligonucleotide with various modifications for pharmacological properties such as RNase protection and enhanced tissue and cell uptake. They differ from normal RNA, for example, in complete sugar 2'-0-methylation, thiophosphate ester sugar binding, eg the cholesterol moiety at the 3'end. In some embodiments, the antago mir is a 2'-O-methyl modification of the molecule at all nucleotides, a cholesterol moiety at the 3'end, and two thiophosphates at the first two positions at the 5'end. It contains an ester sugar bond and four thiophosphate ester bonds at the 3'end. Antago mir can be used to efficiently silence endogenous miRNAs by forming double chains containing antago mirs and endogenous miRNAs, thereby inhibiting miRNA-induced gene silencing. be able to. An example of antago mir-mediated miRN silencing is the silencing of miR-122 described in Krutzfeldt et al., Nature, 2005, 438: 685-689, which is incorporated herein by reference in its entirety.

Modified Single Oligonucleotide Variants Typically bind to cofactors in two components: double-stranded nucleic acid motifs required for recognition by RNAi-related proteins, and RISC's argonaute-catalyzed proteins. A guide strand, which acts as an mRNA, is required to activate the RNAi mechanism. More recently, a novel class of RNAi molecules consisting of one short (25-28 nt) oligo capable of self-polymerizing into partially complementary duplexes has been developed. These molecules have been shown to efficiently activate RISC and produce targeted mRNA silencing comparable to that obtained with predominant conventional RNAi molecules. See, for example, Lapierre et al., “Influential and Systematic RNAi-mediated Silencing with Single Oligonucleotide Compounds” RNA 2011; 17:00, which is incorporated herein by reference in its entirety. Also incorporated herein by reference in its entirety is International Patent Application Publication No. WO 2010/090762 “RNA duplex with single-stranded thiophosphate nucleotide regions for additional functionality” (PCT / See US2010 / 03448).

Accordingly, the present invention includes RNAi constructs containing single-stranded regions of thiophosphate-modified nucleotides, and the use of such constructs in gene silencing.

In some embodiments, the invention utilizes an isolated double-stranded nucleic acid molecule comprising a guide strand and a passenger strand, wherein the passenger strand is one of at least eight thiophosphate-modified nucleotides. It is attached to the chain region by a cleaveable linker. In some embodiments, the invention utilizes an isolated double-stranded nucleic acid molecule comprising a guide strand and a passenger strand, wherein at least one of the guide strand and the passenger strand is at least 6 thiophosphates. It is attached to the single-stranded region of the ester-modified nucleotide by a cleaveable linker.

In some embodiments, the invention utilizes an isolated double-stranded nucleic acid molecule having a guide strand and a passenger strand, wherein at least one of the guide strand and the passenger strand is at least three thiophosphates. It is attached to the single-stranded region of the ester-modified nucleotide by a cleaveable linker. In some embodiments, the double-stranded nucleic acid molecule comprises at least one of the following properties: The passenger chain can be 8-18 nucleotides in length. Nucleic acid can have at least one 2'-O-methyl or 2'-fluoro modification. Cleavable bonds can be other than nucleotide bonds. Nucleic acids may contain lipophilic groups. The guide strand can be 16-18 nucleotides in length or 26-18 nucleotides in length. In some embodiments, the single-stranded region is attached to the guide strand.

In some embodiments, the cleavable bond comprises one or more unmodified nucleotides. In another embodiment, the cleavable linker is a phosphodiester bond. In one embodiment, the cleavable linker is S-S. In some embodiments, the cleavable linker is DNA or RNA. The single-stranded region of at least eight thiophosphate-modified nucleotides may be at either the 3'or 5'end of the passenger chain. In some embodiments, the single-stranded region of at least eight thiophosphate-modified nucleotides is DNA, but in other embodiments RNA.

In some embodiments, the double chain region of the nucleic acid molecule is a complete double chain. In another embodiment, the double-stranded region comprises at least one bulge region. In some embodiments, the passenger chain comprises a nick within the double-stranded region of the molecule. The double-stranded region may contain at least one nucleotide that has been thiophosphate-modified.

Nucleic acid molecules used in accordance with the present invention may be chemically modified. In certain embodiments, the chemical modification is 2'-O-methyl and / or 2'-fluoro. In some embodiments, more than one chemical modification is present on the same molecule. In some embodiments, chemical modification increases stability, increases immune regulatory evasion, and / or inhibits off-target gene silencing. Chemical modifications may be present on the passenger chain and / or the guide chain. In some embodiments, the single-stranded region of at least eight thiophosphate-modified nucleotides is cleaved from the double-stranded region of the nucleic acid molecule in the cell. In some embodiments, the single-stranded region of at least eight thiophosphate-modified nucleotides has complementarity to the mammalian gene. In certain embodiments, the single-stranded region of at least eight thiophosphate-modified nucleotides functions as an antisense molecule. The double-stranded region may be at least 19 nucleotides in length. In some embodiments, the single-stranded region is at least 12 nucleotides in length.

In some embodiments, the invention is a bifunctional nucleic acid molecule comprising a double-stranded region that functions in RNA interference and a single-stranded region that functions in antisense, wherein the double-stranded region is the guide strand. And a bifunctional nucleic acid molecule comprising a passenger strand and the double-stranded and single-stranded regions bound by a cleavable linker is utilized. In some embodiments, the cleavable linker comprises one or more unmodified nucleotides. In another embodiment, the cleavable linker is a phosphodiester bond. In one embodiment, the cleavable linker is S-S. In some embodiments, the cleavable linker is DNA or RNA.

In some embodiments, the invention is a method for inhibiting the expression of a target gene in a mammalian cell, wherein the mammalian cell is subjected to an isolated double-stranded nucleic acid comprising a guide strand and a passenger strand. It relates to a method comprising contacting with a molecule, wherein the passenger chain is attached to a single chain region of at least eight thiophosphate modified nucleotides by a cleavable linker. In some embodiments, the cleavable linker comprises one or more unmodified nucleotides. In another embodiment, the cleavable linker is a phosphodiester bond. In certain embodiments, the cleavable linker is S-S. In some embodiments, the cleavable linker is DNA or RNA.

In some embodiments, the single-stranded region comprises at least 8 thiophosphate-modified nucleotides and may be either the 3'or 5'end of the passenger chain. In some embodiments, the single-stranded region of at least eight thiophosphate-modified nucleotides is DNA, in other embodiments RNA. In some embodiments, the double chain region of the nucleic acid molecule is a complete double chain. In another embodiment, the double-stranded region comprises at least one bulge region. In some embodiments, the passenger chain comprises a nick within the double-stranded region of the molecule. The double-stranded region may contain at least one nucleotide that has been thiophosphate-modified.

Nucleic acid molecules used according to the present invention can be chemically modified. In certain embodiments, the chemical modification is 2'-O-methyl and / or 2'-fluoro. In some embodiments, more than one chemical modification is present in the same molecule. In some embodiments, chemical modification increases stability, increases immune regulatory evasion, and / or inhibits off-target gene silencing. Chemical modifications may be present on the passenger chain and / or the guide chain.

In some embodiments, the single-stranded region of at least eight thiophosphate-modified nucleotides is cleaved from the double-stranded region of the nucleic acid molecule in the cell. In some embodiments, the single-stranded region of at least eight thiophosphate-modified nucleotides has complementarity to the mammalian gene. In certain embodiments, the single-stranded region of at least eight thiophosphate-modified nucleotides functions as an antisense molecule. The double-stranded region may be at least 19 nucleotides in length. In some embodiments, the single-stranded region is at least 12 nucleotides in length.

In some embodiments, the invention is a method for inhibiting the expression of a target gene in a mammalian cell, wherein the mammalian cell is subjected to an isolated double-stranded nucleic acid comprising a guide strand and a passenger strand. It relates to a method comprising contacting with a molecule, wherein the guide strand is attached to a single chain region of at least eight thiophosphate modified nucleotides by a cleaveable linker. In some embodiments, the cleavable linker comprises one or more unmodified nucleotides. In another embodiment, the cleavable linker is a phosphodiester bond. In one embodiment, the cleavable linker is S-S. In some embodiments, the cleavable linker is DNA or RNA.

In some embodiments, the invention is a method of inhibiting the expression of a target gene in a mammalian cell, wherein the mammalian cell functions as a double-stranded region that functions by RNA interference, and antisense. Containing contact with a bifunctional nucleic acid molecule containing a double-stranded region, the double-stranded region comprises a guide strand and a passenger strand, and the double-stranded and single-stranded regions are linked by a cleaveable linker. Yes, about the method. In another aspect, a method of inhibiting the expression of a target gene in a mammalian cell is provided. The method comprises contacting mammalian cells with any of the T-isolated double-stranded nucleic acid molecules described herein.

In some embodiments, the isolated double-stranded nucleic acid molecule used according to the invention comprises a chemical modification that increases stability. In some embodiments, the isolated double-stranded nucleic acid molecule comprises a chemical modification that increases the avoidance of immunoregulation. In some embodiments, the isolated double-stranded nucleic acid molecule comprises a chemical modification that inhibits off-target gene silencing.

In some embodiments, as discussed herein, the oligonucleotide used is a single oligonucleotide molecule that can self-polymerize into a partially complementary double chain. In some embodiments, such single oligonucleotides are approximately 23-30 nucleotides in length, eg, 23, 24, 25, 26, 27, 28, 29 and 30 nucleotides in length. In some embodiments, a single oligonucleotide that can self-polymerize into a partially complementary duplex is about 14-18 nucleotides in the mRNA target region, eg, 14, 15, 16, 17 and 18 nucleotides. Contains the mRNA target region. In some embodiments, a single oligonucleotide that can self-polymerize to a partially complementary double chain can be self-polymerized to a partially complementary double chain, an additional 7-. Includes 11, eg, 7, 8, 9, 10 and 11 nucleotides. In some embodiments, a single oligonucleotide that can self-polymerize into a partially complementary duplex can efficiently enter and activate RNA-induced silencing complex (RISC).

U1 Adapter In some embodiments, the composition provided comprises one or more oligonucleotides that are or act as U1 adapters.

U1 adapters are bifunctional oligonucleotides with a target domain that inhibits the poly A site and is complementary to the site at the target gene exon and a "U1 domain" that binds to the smaller nuclear RNA component of U1 in U1 snRNP. .. See, for example, International Patent Application Publication No. W02008 / 121963 and Goraczniak et al., 2008, Nature Biotechnology, 27 (3), 257-263, each of which is incorporated herein by reference in its entirety. U1 snRNP is a ribonucleoprotein complex that functions primarily to direct early steps in spliceosome formation by binding to the premRNA exon-intron boundary. See Brown and Simpson, 1998, Annu Rev Plant Physiol Plant MoI Biol 49: 77-95.

In some embodiments, the oligonucleotide utilized is a U1 adapter, wherein the oligonucleotide comprises at least one annealing domain (target domain) linked to at least one effector domain (U1 domain), said annealing. A U1 adapter in which a domain hybridizes to a target gene sequence and the effector domain hybridizes to U1 snRNA of U1 snRNP. In some embodiments, the U1 adapter comprises one annealing domain. In some embodiments, the U1 adapter comprises one effector domain.

Without being bound by theory, the annealing domain is typically about 10 to about 50 nucleotides in length, more typically about 10 to about 30 nucleotides in length, or about 10 to about 20 nucleotides in length. In some embodiments, the annealing domain is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides in length. The annealing domain can be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least about 100% complementary to the target gene. In some embodiments, the annealing domain hybridizes to a terminal coding region (eg, via a polyadenylation site) and a target site within the Y-terminal exon of the premRNA containing the 3'UTR and polyadenylation signal sequences. .. In some embodiments, the target site is within about 500 base pairs, about 250 base pairs, about 100 base pairs, or about 50 base pairs of the poly (A) signal sequence of the premRNA. In some embodiments, the annealing domain has one, two, three or four mismatches with the target gene sequence.

In some embodiments, the effector domain ranges from about 8 nucleotides to about 30 nucleotides in length. In some embodiments, the effector domain ranges from about 10 nucleotides to about 20 nucleotides in length. In some embodiments, the effector domain ranges from about 10 nucleotides to about 15 nucleotides in length. The U1 domain can hybridize to U1 snRNA, especially the 5'end, more specifically nucleotides 2-11. In some embodiments, the U1 domain is fully complementary to nucleotides 2-11 of the endogenous U1 snRNA. In some embodiments, the U1 domain is SEQ ID NO: 1 (5'-GCCAGGIL1AAGUAU-3'), SEQ ID NO: 2 (5 "-CCAGGLIAA.GUAII-3"), SEQ ID NO: 4 (5 "-CAGGUAAGUAU-3'). ), SEQ ID NO: 5 (5 ² -CAGGLIAAGU-3'), SEQ ID NO: 6 (5'--CM.16ITAAC1-3'), and SEQ ID NO: 7 (5'-CAGG1IA2 =',, 3'). Containing a nucleotide sequence of choice. In some embodiments, the III domain comprises a nucleotide sequence, SEQ ID NO: 8 (5 "-CAGGUAAGUA-3"), which is not bound by theory, but is a base pair. Increasing the length of the U1 domain to include formation in stem 1 and / or base pairing at position 1 of U1 snRNA improves the affinity of the U1 adapter for U1 snRNA.

The annealing and effector domains of the U1 adapter are linked so that the effector domain is at the 5'end and / or 3'end of the annealing domain. Whether these two domains have the 3'end of one domain linked to the 5'end of the other domain or the 3'end of one domain to the 3'end of the other domain. Alternatively, it can be linked as if the 5'end of one domain is linked to the 5'end of the other domain. Annealing and effector domains can be linked directly to each other, or can be linked by a nucleotide or non-nucleotide linker. In some embodiments, the linker is a nucleotide base and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15, up to 20, or up to 25 nucleotides.

In some embodiments, the linker for the annealing domain and the effector domain is multivalent, eg trivalent, tetravalent, or pentavalent. Without being bound by theory, multivalent linkers can be used to bind a single annealing domain together with multiple adapter domains.

In some embodiments, the U1 adapter comprises any oligonucleotide modification described herein. Exemplary such modifications include modifications that increase annealing affinity, specificity, bioavailability, cell and / or nuclear transport, stability, and / or degradation resistance in cells and organisms. In some embodiments, the U1 adapter may be administered in combination with at least one other RNAi agent.

RNA Activator In some embodiments, the composition provided comprises an RNA activator or one or more oligonucleotides acting as it.

Recent studies have found that dsRNA can also activate gene expression, a mechanism called "small RNA-induced gene activation" or RNAa. For example, Li, LC et al. Proc Nati Auld Sci US A. (2006), 103 (46): 17337-42, and Li LC (2008) "Small RNA-mediated gene expression" RNA and regulation of gene expression: concealment of complexity See Caister Academic Press. ISBN 978-1-904455-25-7. The dsRNA target gene promoter has been shown to induce potent transcriptional activity of related genes. Endogenous miRNAs that cause RNAa have also been found in humans. Check E. Nature (2007) 448 (7156): 855-858.

Another observation is that RNAa gene activity persists for extended periods of time. Induction of gene expression is thought to last for more than 10 days. This long-term effect of RNAa may be due to epigenetic changes at the dsRNA target site.

In some embodiments, the oligonucleotide provided is an RNA activator in which the oligonucleotide provided increases gene expression. In some embodiments, increased gene expression inhibits viability, growth, and / or regeneration.

Non-coding RNA (ncRNA)
The present invention includes non-coding RNAs such as long non-coding RNAs (lncRNAs) and short non-coding RNAs (sncRNAs).

Therefore, the oligonucleotide compositions of the invention provided may be useful for regulating disease-related long non-coding RNAs. In some embodiments, oligonucleotides synthesized according to the methods provided herein specifically block the binding of the transcriptional repressor complex in order to target the lncRNA region, thereby relating to the relevant target. It is used to induce gene expression. In some embodiments, the transcriptional repressor complex is a silencing factor. In some embodiments, the transcriptional repressor complex has histone methyltransferase activity. In some embodiments, the transcriptional repressor complex methylates histone H3. In some embodiments, the transcriptional repressor complex is PRC2 (Polycomb Repressive Complex 2).

Certain PRC2-related lncRNAs have been reported as potential therapeutic targets and / or biomarkers (Zhao et al., 2010. “Genome Identification of Polycomb-Related RNAs by RIP Sequence” Molelcular Cell 40: 939-53). Overexpression of the PCR2 protein has been associated with various types of cancer, including metastatic prostate and breast cancer, and cancers of the colon, milk and liver. Pharmacological inhibition of PRC2-mediated gene suppression was found to induce apoptosis in several cancer cell lines in vitro, but not in various types of normal cells. The induction of apoptosis in this system relies on the reactivation of genes suppressed by PRC2. There is also evidence that PRC2-mediated gene suppression may be associated with the maintenance of stem cell properties in cancer stem cells. This result shows that, at least in some cases, inhibition of PRC2-mediated gene suppression, including by targeting lncRNAs that rescue PRC2 to important genes, is predominant for treating various types of cancer. It suggests that it is a strategy. Non-limiting sequences of nucleic acids that can be prepared according to the methods provided herein are, for example, WO 2012, International Patent Application Publication, entitled "Polycomb-Related Non-coding RNA", the contents of which are incorporated herein by reference. It is found in the 065143 issue (PCT / US2011 / 60493).

The oligonucleotide compositions provided by the present invention may or may work as small non-coding RNAs such as piwi-interacting RNAs (piRNAs). PiRNA is the largest type of small non-coding RNA molecule expressed in animal cells and is found in clusters in the genome. piRNA is known to form an RNA-protein complex by interacting with piwi proteins. PiRNA complexes are associated with both retrotransposon metamorphic gene silencing and post-transcription gene silencing, as well as other genetic elements in germ cells, including spermatogenesis. Typically, piRNAs are 26-31 nucleotides in length, lack sequence conservation and exhibit higher complexity compared to typical miRNAs. In some embodiments, the oligonucleotide composition utilized in accordance with the present invention comprises 5'uridine. In some embodiments, the oligonucleotide composition utilized in accordance with the present invention comprises a 5'monophosphate and a 3'modification that acts to block either 2'or 3'oxygen. In some embodiments, the 3'modification is 2'-O-methylation.

Triple-strand-forming oligonucleotides In some embodiments, the composition provided comprises one or more oligonucleotides that are or serve as triple-strand-forming oligonucleotides.

Recent studies have shown that triple-strand-forming oligonucleotides (TFOs) that can recognize and bind polypurine and / or polypyrimidine regions of double-stranded helical DNA in a sequence-specific manner can be designed. These laws of recognition are Maher III, LJ et al., Science (1989) vol. 245, pp 725-730; Moser, HE et al., Science (1987) vol. 238, pp 645- 630; Beal, PA et al., Science. (1992) vol. 251, pp 1360-1363; Conney, M. et al., Science (1988) vol. 241, pp 456-459, and Hogan, ME et al., EP Publication No. 375408. Oligonucleotide modifications, such as the introduction of interchilators, intersaccharide binding substitutions, and optimization of binding conditions (pH and cation concentrations), help eliminate inherent obstacles to TFO activity such as charge repulsion and instability. It has recently been shown that oligonucleotides can target specific sequences (see Seidman and Glazer, Clin Invest 2003 2.: 487-94 for a recent review). In general, triple-strand-forming oligonucleotides have the following sequence match:
Oligo 3'-AGGT
Double chain 5'-AGCT
Double Chain 3'-TCGA

However, the A-AT and G-GC triple chains have been shown to have maximum triple helix stability (Reither and Jeltsch, BMC Biochem, 2002, Sept12, Epub). The same authors demonstrate that TFOs designed according to the A-AT and G-GC rules do not form non-specific triple chains. The triple chain formation is shown to be sequence-specific in nature. Therefore, for any given sequence, a triple chain forming sequence can be devised. In some embodiments, the triple chain-forming oligonucleotide is at least about 15, about 25, or about 30 or more nucleotides in length. In some embodiments, the triple chain forming oligonucleotide is up to about 50 or about 100 nucleotides in length.

The formation of a triple-stranded helical structure with the target DNA induces steric and functional changes, blocks transcription initiation and elongation, introduces desired sequence changes in endogenous DNA, and specifically downregulates gene expression. Bring. An example of such suppression of gene expression in TFO-treated cells induces knockacts of episome supFGI and endogenous HPRT in mammalian cells (Vasquez et al., Nucl Acids Res. 1999; 27: 1176- 81, and Puri et al., J Biol Chem, 2001; 276: 28991-98), Ets2 transcription factor (Carbone et al., Nucl Acid Res. 2003; 31: 833-43) and preinflammatory ICAM-I gene (Besch et al., Sequences of expression of J Biol Chem, 2002; 277: 32473-79)-and target-specific downregulation are important in prostate cancer epidemiology. Furthermore, Vuyisich and Beal have recently shown that sequence-specific TFOs can bind to dsRNA and inhibit the activity of dsRNA-dependent enzymes such as RNA-dependent kinases (Vuyisich and Beal, Nuc, Acids Res 2000; 28). : 2369-74).

In addition, TFOs designed according to the above principles can induce directional mutations that can achieve DNA repair, thus providing down-regulation and up-regulation of endogenous gene expression (Seidman and Glazer, JInvest 2003; 112: 487-94). A detailed description of the design, synthesis and administration of effective TFOs is incorporated herein by reference in its entirety, Froehier et al. S. Pat. App. Nos. 2003 017068 and 2003 0096980, and Emanude et al. It is found in 2002 0128 21/8 and 2002 0123476, as well as in Lawn's US Pat. No. 5,721,138.

Complex / Linker The invention also considers that, in some embodiments, the oligonucleotide utilized is optimized for cellular uptake. In any utilized oligonucleotide encapsulated by the present invention, the guide and / or passenger chain may be attached to the complex. In some embodiments, the complex is hydrophobic. The hydrophobic complex may be a small molecule with a partition coefficient greater than 10. Complexes are sterol-type molecules, such as cholesterol, or molecules with increased length polycarbon chains bound to C17, and the presence of the complex is RNA taken up by cells with or without lipid transfection reagents. It can affect the ability of the molecule. The complex can be attached to the passenger or guide chain by a hydrophobic linker. In some embodiments, the hydrophobic linker is 5-12 C long and / or hydroxypyrrolidine type. In some embodiments, the hydrophobic complex is attached to the passenger and the CU residue of either the passenger and / or the guide chain is modified. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the CU residues on the passenger and / or guide strand are , Qualified. In some embodiments, the molecules associated with the invention are self-delivering (sd). As used herein, "self-delivery" means the ability of a molecule to be delivered to a cell without the need for additional delivery vehicles such as transfection reagents. Aspects of the invention relate to selecting molecules for use in RNAi.

In any of the embodiments incorporated herein, the oligonucleotide utilized may associate with a hydrophobic moiety for targeting and / or delivering the molecule to the cell. In some embodiments, the hydrophobic moiety is associated with the nucleic acid molecule via a linker. In some embodiments, the association is by a non-covalent interaction. In some embodiments, the meetings are covalently bonded. Any linker known in the art can be used to associate with nucleic acids having hydrophobic moieties. Linkers known in the art are published PCT applications, WO 92/03464, WO 95/23162, WO 2008 /, which are incorporated herein by reference. 021157, WO 2009/021157, WO 2009/134487, WO 2009/126933, U.S. Patent Application Publication No. 2005/0107325, U.S. Patent No. 5,414,077, No. 5,419,966, No. It is described in No. 5,512,667, No. 5,646,126, and No. 5,652,359. The linker may be as simple as a covalent bond to a polyatomic linker. The linker may be cyclic or non-cyclic. The linker may be optionally substituted. In some embodiments, the linker can be cleaved from nucleic acid. In certain embodiments, the linker can be hydrolyzed under physiological conditions. In some embodiments, the linker can be cleaved by an enzyme (eg, esterase or phosphodiesterase). In some embodiments, the linker comprises a spacer element for separating nucleic acid from the hydrophobic moiety. The spacer element may contain 1-30 carbon atoms or heteroatoms. In certain embodiments, the linker and / or spacer element comprises a protonatable functional group. Such protonatable functional groups can promote endosome escape of nucleic acid molecules. Protonable functional groups also facilitate the delivery of nucleic acids to cells, eg, neutralize the overall charge of the molecule. In some embodiments, the linker and / or spacer element is biologically inactive (ie, does not impart biological activity or function to the resulting nucleic acid molecule).

Hydrophobic molecules can be attached to polynucleotides by linker molecules. Optionally, the linker moiety is a non-nucleotide linker moiety. Non-nucleotide linkers are, for example, debase residues (sSpacer), oligoethylene glycols such as triethylene glycol (spacer 9) or hexaethylene glycol (spacer 18), or alkanediols such as butanediol. Spacer units are preferably linked by phosphodiester or thiophosphate bonds. The linker unit may appear only once in the molecule or may be incorporated several times, for example by phosphodiester, thiophosphate, methylphosphate or amide bonds.

A typical complexed protocol involves the synthesis of a polynucleotide having an aminolinker at one or more positions in the sequence, but no linker is required. The amino group then reacts with the complexed molecule using a suitable coupling or activation reagent. The complexing reaction can be accomplished either with the polynucleotide as is bound to the solid support or by following cleavage of the polynucleotide in the solution phase. Purification of modified polynucleotides by HPLC typically yields pure material.

In some embodiments, the linking group can be attached to the nucleomonomer and the transport peptide can be covalently attached to the linker. In some embodiments, the linker may function as the binding site for both of the transport peptides and may provide stability to the nuclease. Examples of suitable linkers are substituted or unsubstituted C ₁ -C ₂₀ alkyl chains, C ₂ -C ₂₀ alkenyl chains, C ₂ -C ₂₀ alkynyl chains, peptides, and heteroatoms (eg, S, O, NH, etc.). Including. Other exemplary linkers include bifunctional crosslinkers such as sulfosuccinimidyl-4- (maleimidephenyl) -butyrate (SMPB) (see, eg, Smith et al. Biochem J 1991. 276: 417-2). ).

In some embodiments, the oligonucleotides provided by the invention are synthesized as molecular complexes that utilize a receptor-mediated endocytosis mechanism for delivering genes to cells (eg, Bunnell et al. 1992. Somatic). See Cell and Molecular Genetics. 18: 559, and references cited herein).

Targeting Agent In some embodiments, the provided composition comprises one or more targeting agents associated with the composition and / or the individual oligonucleotides contained therein. Examples of such targeting agents are described above and further described herein. The terms "targeting agent" and "targeting moiety" are used interchangeably herein.

Delivery of oligonucleotides and / or compositions thereof can generally be improved by delivering oligonucleotides to cell receptors. The targeting moiety can be complexed to an oligonucleotide or attached to a carrier group linked to the oligonucleotide (eg, poly (L-lysine) or liposome). The method is well adapted to cells exhibiting specific receptor-mediated endocytosis.

For example, oligonucleotide complexes for 6-phosphomannosylated proteins are uptaken 20-fold more efficiently by cells expressing mannose 6-phosphate-specific receptors than free oligonucleotides. Oligonucleotides can also be attached to ligands for cell receptors using biodegradable linkers. In another example, the delivery construct is a mannosylated streptavidin that forms a tight complex with a biotinylated oligonucleotide. Mannosylated streptavidin has been shown to increase the internalization of biotinylated oligonucleotides 20-fold (Vlassov et al. 1994. Biochimica et Biophysica Acta 1197: 95-108).

The field of RNA interference has revolutionized the study of biological processes. These tools include small molecule inhibitory RNA (siRNA), small molecule hairpin RNA (shRNA), and ribozymes. As RNA interference technology advances, so does the list of confirmed RNA target sequences and reagents for use. The provided compositions and methods encapsulated in this application can be readily applied to each of such target sequences. See, for example, http://www.rnainterference.org/index.html for a database of proven siRNA target sequences and links to kits and reagents that can be used for RNA interference experiments. Exemplary siRNA target sequences useful in the present invention are provided in Attachment (A).

Immunomodulatory Oligonucleotides In some embodiments, the composition provided comprises one or more oligonucleotides that are or act as immunomodulatory oligonucleotides.

Oligonucleotides used in accordance with the present invention can be immunomodulators, ie substances that can regulate or control an immune response when administered to a subject. The immune response elicited by such a substance can be an innate and / or compatible immune response. The immune system is divided into a more innate immune system and an acquired compatible immune system in vertebrates. The latter of these is further classified into humoral cell components. In some embodiments, the immune response may be mucosal. The immunomodulatory motifs described herein can be used in the context of the classes described above for immunomodulatory oligonucleotides including ODN classes such as A class, B class, C class, E class, T class and P class. ..

In some embodiments of the invention, the immunomodulatory oligonucleotides used according to the invention comprise one or more immunomodulatory motifs. In some embodiments, oligonucleotides used in accordance with the present invention include one or more "CpG dinucleotides". CpG dinucleotides may or may not be methylated. The immunostimulatory oligonucleotide containing at least one unmethylated CpG dinucleotide is an unmethylated cytosine-guanine dinucleotide sequence (ie, unmethylated 5'cytidine, followed by 3'guanosine, and a phosphate ester. It is an oligonucleotide molecule that contains (linked by binding) and activates the immune system; such immunostimulatory oligonucleotides are CpG oligonucleotides. CpG oligonucleotides include U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and 6,339,068, the contents of which are incorporated herein by reference. , Numerous issued patents, published patent applications, and other publications. In some embodiments, the oligonucleotide used is or acts as an agonist of a Toll-like receptor. In some embodiments, the oligonucleotide used is or acts as an agonist of Toll-like receptor 9 (TLR9). In some embodiments, the oligonucleotide used is or acts as an antagonist of a Toll-like receptor. In some embodiments, the oligonucleotide used is or acts as an antagonist of Toll-like receptor 9 (TLR9). In some embodiments, the sterically identified oligonucleotides included by the present invention show higher affinity for each receptor / target compared to their random counterparts. In some embodiments, the sterically identified oligonucleotides included in the invention are administered to subjects who meet certain clinical criteria with less degree of variation between individuals compared to their random steric counterparts. When done, it induces one or more immune responses. In some embodiments, the steric identified oligonucleotides contained by the present invention have less toxic side effects when administered to subjects who meet clinical criteria as compared to their random counterparts. Causes or causes few side effects.

In some embodiments, the immunostimulatory oligonucleotides used according to the invention do not have a CpG dinucleotide motif. These oligonucleotides that do not have a CpG dinucleotide motif are referred to as non-CpG oligonucleotides and have a non-CpG immunostimulatory motif. In some embodiments, these are T-rich immunostimulating oligonucleotides, eg, oligonucleotides with at least 80% T.

In some embodiments, the immunostimulatory oligonucleotide used according to the present invention is a class B immunomodulatory oligonucleotide.

"B-class" ODNs are potent in B cell activation but relatively weak in inducing IFN-α and NK cell activation. B-class CpG oligonucleotides are typically well stabilized and contain unmethylated CpG dinucleotides within certain preferred base circumstances. See, for example, U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and 6,339,068.

Another class is potent in IFN-α and NK cell activation, but relatively weak in stimulating B cells; this class is referred to as the "A class". In some embodiments, the immunostimulatory oligonucleotide used according to the present invention is a class A immunomodulatory oligonucleotide. Class A CpG oligonucleotides typically contain a stabilized polyG sequence at the 5'and 3'ends, and a parindrome phosphodiester CpG dinucleotide sequence at least 6 nucleotides. See, for example, the published patent application PCT / US00 / 26527 (WO 01/22990).

Yet another class of CpG oligonucleotides activates B and NK cells and induces IFN-α; this class is called the C class. In some embodiments, the immunostimulatory oligonucleotide used in accordance with the present invention is a class C immunomodulatory oligonucleotide. "C-class" immunostimulatory oligonucleotides contain at least two different motifs and have a unique and desirable stimulatory effect on cells of the immune system. Some of these ODNs have both a typical "stimulating" CpG sequence and a "GC-rich" or "B cell neutralization" motif. These combination motif oligonucleotides have immunostimulatory effects associated with typical "class B" CpG ODN, which are potent inducers of B cell activation and dendritic cell (DC) activation, and IFN-α and A more recently described tail class of immunostimulatory oligonucleotides (“Class A” CpG ODN), a potent inducer of natural killer (NK) cell activation but a relatively weak inducer of B cell and DC activation. It has an immunostimulatory effect, which is contained somewhere between the immunostimulatory effects associated with. Krieg AM et al. (1995) Nature 374: 546-9; Ballas ZK et al. (1996) J Immunol 157: 1840-5; Yamamoto S et al. (1992) J Immunol 48: 407 2-6. The preferred class B CpG ODN often has a thiophosphate ester skeleton, the preferred class A CpG ODN has a mixed or chimeric skeleton, but the C class of combination motif immunostimulating oligonucleotides is stabilized, eg. It may have either a thiophosphate, chimeric, or phosphodiester skeleton, and in some embodiments has a quasi-soft skeleton. This class is described in US Patent Application No. USI 0 / 224,523, filed August 19, 2002, the entire contents of which are incorporated herein by reference.

In some embodiments, the immunostimulatory oligonucleotide used according to the present invention is an E-class immunomodulatory oligonucleotide. "E-class" oligonucleotides have an enhanced ability to induce the secretion of IFN-α. These ODNs have lipophilic substituted nucleotide analogs 5'and / or 3'in the YGZ motif. Compounds of E-class formula have, for example, one of the following lipophilic substituted nucleotide analogs: substituted pyrimidine, substituted uracil, hydrophobic T analog, substituted toluene, substituted imidazole or pyrazole, substituted triazole, 5-chloro-uracil, 5-bromo-uracil, 5-iodo-uracil, 5-ethyl-uracil, 5-propyl-uracil, 5-propynyl-uracil, (E) -5- (2-bromovinyl) -uracil, or 2,4-difluoro -toluene. E-class oligonucleotides are described at least in US Provisional Application No. 60 / 847,811.

In some embodiments, the immunostimulatory oligonucleotide used according to the invention is a T-class immunomodulatory oligonucleotide. "T-class" oligonucleotides, when unmodified as in the ODN, IFN-related cytokines and chemokines of the invention, induce lower levels of IFN-α secretion than B or C-class oligonucleotides at the same time. Retains the ability to induce IL-10 levels similar to B-class oligonucleotides. T-class oligonucleotides are described, at a minimum, in US Patent Application No. 11 / 099,683, the entire contents of which are incorporated herein by reference.

In some embodiments, the immunostimulatory oligonucleotide used according to the present invention is a P-class immunomodulatory oligonucleotide. A "P-class" immunomodulatory oligonucleotide has several domains, including a 5TLR activation domain, a double double chain formation region and any spacers and a 3'tail. In some cases, this type of oligonucleotide has the ability to induce much higher levels of IFN-α than C-class. P-class oligonucleotides have the ability to spontaneously self-assemble and become concatomers either in vitro and / in vivo. Although not bound by any particular theory as to how these molecules act, one possible hypothesis is that this property has the ability to more highly cross-link to TLR9 within a particular immune cell. It is to provide a class oligonucleotide and induce a distinctly different pattern of immune activation compared to the class of CpG oligonucleotides described above. Cross-linking of TLR9 receptors can induce stronger IFN-α activation by the type I IFNR feedback loop in plasmacytoid dendritic cells. P-class oligonucleotides are described at least in US Patent Application No. 11 / 706,561.

In some embodiments, the immunostimulatory oligonucleotide used according to the present invention is an S-class immunomodulatory oligonucleotide. The immunomodulatory oligonucleotides of the present invention can be immunosuppressive oligonucleotides. The immunomodulatory motifs described herein can be used in the context of the above classes of immunosuppressive oligonucleotides, including ODN classes such as "S class". Inhibition, or S-class ODN, is useful whenever desired to inhibit immune stimulation. Inhibiting ODNs include infectious shock, inflammation, allergies, asthma, transplant rejection, transplant-to-host disease (GvHD), autoimmune diseases, Th1- or Th2-intervening diseases, bacterial infections, parasite infections, spontaneous absorption, And can be used to prevent and treat tumors. Inhibiting ODNs generally inhibit the activation of all cells expressing the associated TLR, more specifically antigen-presenting cells, B cells, plasmacytoid dendritic cells (pDCs), monocytes, It can be used to inhibit the activation of monocyte-derived cells, eosinophils, and neutrophils. S-Class ODN is further described in at least US Patent Application No. 10 / 977,560.

According to the present invention, the immunomodulatory oligonucleotide may have a skeleton of stabilized internucleotide linkage or a chimeric skeleton of stabilized phosphodiester nucleotide linkage in addition to the stabilized FANA purine nucleotides (s). "Stabilized internucleotide binding" is intended to mean an internucleotide binding that is relatively resistant to in vivo degradation (eg, by exonuclease or endonuclease) as compared to phosphodiester nucleotide binding. In some embodiments, the stabilized internucleotide bonds are thiophosphate, dithiophosphate, methylphosphate, thiomethylphosphate, phosphonoacetate, Rp-thiophosphate, Sp-thiophosphate, boranophosphate, Or include, but is not limited to, 3'-thioformacetal, or mixtures thereof. Other stabilized oligonucleotides include: nonionic DNA analogs such as alkyl and aryl phosphates (charged phosphate esters in which oxygen is replaced by alkyl or aryl groups), phosphodiesters, and charged oxygen moieties. Alkyl phosphodiester that is alkylated. Oligonucleotides containing diols, such as tetraethylene glycol or hexaethylene glycol at either or both ends, have also been found to be substantially resistant to nuclease degradation.

As described in more detail herein, according to the invention, these and other modifications are predetermined positions within the oligonucleotide to obtain a stereospecific oligonucleotide molecule. ) / Patterns can be selectively introduced. Moreover, according to the invention, compositions containing a large number of such oligonucleotides are of fairly high purity (eg, at least 96%, at least 97%, at least 98%, at least 99%, essentially 100%). Obtained at. Therefore, provided oligonucleotide compositions comprising one or more predetermined types of oligonucleotides are suitable for in vivo administration. Due to the high structural purity of the oligonucleotides in the composition, thereby the composition comprises one particular type of oligonucleotide, the provided composition is improved when administered to the subject. It can exhibit increased biological activity, increased efficiency, reduced response variables, and / or reduced unwanted side effects.

Therefore, the oligonucleotide used in the present invention is specifically useful as a therapeutic agent when it is formulated into a pharmaceutical composition. In some embodiments, such therapeutic agents are immunomodulators. In some embodiments, the immunomodulatory agent provided is an immunostimulatory agent. In some embodiments, the immunomodulatory agent provided is an immunosuppressive (or immunosuppressive) agent. In some embodiments, the immunomodulatory agent provided acts as an adjuvant. In some embodiments, the immunomodulator provided can turn a Th2 immune response into a Th1 immune response by inducing a Th1 cytokine that can suppress the inflammatory Th2 response. In some embodiments, the oligonucleotide compositions provided are useful as substances that control gene expression. For example, in some embodiments, the oligonucleotide composition provided is useful as a substance capable of silencing a gene of interest, eg, a gene associated with a disease or disorder. In some embodiments, the oligonucleotide compositions provided are useful as substances for controlling RNA splicing.

For example, embodiments of such oligonucleotides include those comprising at least one CpG dinucleotide motif. In some embodiments, such oligonucleotides contain at least one unmethylated CpG dinucleotide motif. In some embodiments, CpG-containing oligonucleotides useful in the present invention are classified as Class A (Class D) oligonucleotides. In some embodiments, CpG-containing oligonucleotides useful in the present invention are classified as Class B (Class K) oligonucleotides. In some embodiments, CpG-containing oligonucleotides useful in the present invention are classified as Class C oligonucleotides. In some embodiments, CpG-containing oligonucleotides useful in the present invention are classified as Class P oligonucleotides. In some embodiments, the CpG-containing oligonucleotide useful in the present invention comprises at least one palindrome sequence. In some embodiments, the CpG-containing oligonucleotides useful in the present invention may form a "dumbbell-like" structure. In some embodiments, the CpG-containing oligonucleotides useful in the present invention may form a "Y" -like structure or a multimer thereof. In some embodiments, the CpG-containing oligonucleotides useful in the present invention may form a tetrahedral structure. For a review, see, for example, Bode et al. (2011) Expert Rev. Vaccines 10 (4): 499-511; Hanagata (2012) Int. J. Nanomedicine 7: 2181-95.

In some embodiments, oligonucleotides used according to the invention elicit immunomodulatory effects on cells expressing Toll-like receptors. In some embodiments, the target cells that respond to such oligonucleotides are antigen presenting cells (APCs), antigen-specific T and B cells, cytotoxic T cells (CTLs), natural killer (NK). Includes, but is not limited to, cells and dendritic cells (DCs). In some embodiments, the immunomodulatory effect is a direct effect elicited by a cell that expresses a receptor that recognizes such oligonucleotides or a plurality of receptors. In some embodiments, such oligonucleotides elicit immunomodulatory effects on cells expressing Toll-like receptors 9 (TLR9) as ligands. For example, some embodiments of the invention include CpG oligonucleotides that act as agonists of one or more Toll-like receptors. Some embodiments of the invention include CpG oligonucleotides that act as antagonists of one or more Toll-like receptors. In some embodiments, the immunomodulatory effect is an indirect effect that occurs downstream, including cells that do not necessarily respond directly to such oligonucleotides or express such receptors.

In some embodiments, the CpG oligonucleotides useful for the present invention are characterized by the direct activation of human B cells and plasmacytoid dendritic cells by TLR-9. In some embodiments, CpG oligonucleotides useful for the present invention are characterized by indirectly supporting the maturation and proliferation of natural killer cells, T cells and monocytes / macrophages.

In some embodiments, the CpG oligonucleotides useful for the present invention are characterized by the production of Th1 type and pre-inflammatory cytokines, chemokines and polyreactive IgM.

In some embodiments, CpG oligonucleotides useful for the present invention are characterized in that the immunogenicity of conventional protein antigens and peptide-type vaccines is enhanced by such oligonucleotides. Without being bound by a particular theory, such an adjuvant effect is mediated by the improved function of specialized antigen-presenting cells and the generation of humoral and cell-mediated vaccine-specific immune responses. It is considered.

In some embodiments, CpG oligonucleotides useful for the present invention are characterized by the CpG oligonucleotide increasing the extent of the vaccine-induced response and facilitating the progression of the response. They also improve the induction of memory, thereby prolonging the duration of humoral and cell-mediated immunity.

In some embodiments, CpG oligonucleotides useful for the present invention enhance the immunity of a group of subjects with reduced immune function (eg, a patient population), such as an elderly person and a person with an inhibitory immune system. It is characterized by. These are effective when administered either systemically or mucosally. Preclinical and clinical trials with mixed chirality (eg, not chirally pure) CpG oligonucleotides can enhance the immunogenicity of vaccines targeting infectious diseases and cancers. In addition, clinical trials point out that CpG oligonucleotides are reasonably safe when administered as a vaccine adjuvant.

Immunomodulatory oligonucleotides prepared according to the disclosure of the present invention are useful for a number of therapeutic applications. Therefore, the immunomodulatory oligonucleotide used can be formulated into a suitable pharmaceutical composition. Such pharmaceutical compositions may be administered to a subject in an amount effective for treating a disease, disorder or symptom as further described herein by a suitable route of administration. According to the present invention, an effective amount of an immunostimulating oligonucleotide can be administered to a subject who is likely to benefit from enhancing the immune system (eg, an enhanced immune response). In some embodiments, clinical benefits are conferred directly by immunomodulatory oligonucleotides that act on their target immune cells, eg, by binding a ligand to their Toll-like receptor protein. In some embodiments, clinical benefits are achieved, at least in part, indirectly by enhancing the subject's immune system overall.

As used herein, the terms "effective amount" and "effective dosage" are the intended purpose (s), that is, the desired biological or pharmaceutical response in a tissue or subject with an acceptable benefit / risk ratio. Refers to any amount or dosage of compound or composition sufficient to fill. Appropriate intended purpose may be objective (ie, measurable by a test or marker) or subjective (ie, the subject shows signs of effect or feels effect). In some embodiments, the therapeutically effective amount meets specific clinical criteria for the disease or disorder (eg, as determined by manifested symptoms, disease progression / stage, genetic profile, etc.). When administered to a subject group, a statistically significant therapeutic response is obtained in the population. Therapeutically effective doses are generally administered in a dosing regimen that may include multiple unit dosages. For any particular pharmaceutical substance, a therapeutically effective amount (and / or a suitable unit dosage within an effective dosing regimen) may vary, for example, depending on the route of administration combined with the other pharmaceutical substance. In some embodiments, the specific therapeutically effective amount (and / or unit dosage) for any particular patient is the disease to be treated and the severity of the disease; of the specific pharmaceutical substance employed. Activity; specific composition adopted; patient's age, weight, general health, gender and nutrition; time of administration adopted, route of administration, and / or specific pharmaceutical substance excretion and metabolic rate; duration of treatment. It can rely on a variety of factors, including as well as similar factors well known in the pharmaceutical field. Those skilled in the art will consider that in some embodiments of the invention, an effective amount is included where the single or position dosage comprises a suitable dose for administration in the context of a dosing regimen associated with a positive result. Thus, in some embodiments, CpG oligonucleotides prepared according to the present invention may exhibit improved efficacy and immunomodulatory effects due to their chiral purity. In some embodiments, the effective amount of immunomodulatory oligonucleotide provided is lower than that of the less pure counterpart.

In some embodiments, the immunomodulatory oligonucleotide composition is administered to a healthy subject. In some embodiments, the immunomodulatory oligonucleotide composition is administered to the subject as part of a vaccine, the immunomodulatory oligonucleotide acting as an adjuvant. In some embodiments, the immunomodulatory oligonucleotide composition is administered to the subject in a suppressed (eg, damaged) immune system. In some embodiments, subjects whose immune system is suppressed and who do not respond adequately to conventional vaccines respond to vaccines comprising immunomodulatory oligonucleotides encapsulated by the present invention. In some embodiments, subjects who may benefit from such vaccines suppress the immune system associated with infectious diseases such as viral infections such as HIV, HBV, HCV and the like.

The present invention includes the use of immunomodulatory oligonucleotides contemplated herein for the treatment of subjects with symptoms that can be treated by stimulating or suppressing an immune response. Accordingly, the immunomodulatory oligonucleotide compositions provided are for the treatment of diseases or symptoms including, but not limited to, infectious diseases, cancers, allergies, asthma, inflammatory symptoms, autoimmune diseases, and any combination thereof. It is useful.

In some embodiments, the immunomodulatory oligonucleotide compositions provided are useful in certain embodiments of the invention for the treatment of subjects at risk of developing clinical symptoms. Non-limiting clinical manifestations include allergies, asthma, infectious organism infections, inflammation and autoimmune diseases. A subject at risk as used herein is a subject who has any risk of exposure to an infection that causes a pathogen, cancer or allergen, or is at risk of developing cancer. For example, a subject at risk may be a subject planning a trip to an area where a particular type of infectious agent is found, and a body fluid that may contain an infectious organism through its lifestyle or through medical means. Can be a subject exposed to or directly exposed to the organism, or even any subject living in an area where an infectious organism or allergen has been identified. Subjects at risk of developing an infectious disease also include the general population for which medical institutions recommend vaccination with certain infectious bioantigens. If the antigen is an allergen and the subject develops an allergic reaction to the particular antigen and the subject can be exposed to that antigen or in the pollen season, then the subject is at risk of exposure to that antigen. .. Subjects at risk of developing allergies or asthma are those identified as having allergies or asthma but not having active disease during immunomodulatory oligonucleotide treatment, and for general or environmental factors. Includes subjects who are considered to be at risk of developing the disease. Subjects at risk of developing cancer are those who have a high probability of developing cancer. These subjects are, for example, those with systemic abnormalities whose presence has been shown to correlate with a higher likelihood of developing cancer, and tobacco, asbestos or other chemistries. Subjects who have been exposed to cancer-causing substances such as toxins, or who have been treated for cancer and are clearly in remission. When a subject at risk of developing cancer is treated with an antigen specific to the cancer type at risk of developing the subject and a CpG immunostimulatory oligonucleotide, the subject kills the cancer cell as it develops. obtain. If the tumor begins to form in the subject, the subject will develop a specific immune response to the tumor antigen.

In some embodiments, the immunomodulatory oligonucleotide compositions provided are useful for treating subjects with immune disorders or disorders, including asthma, allergies and related symptoms.

A subject with an allergy is a subject at or at risk of developing an allergic reaction in response to an allergen. Allergy refers to acquisition hypersensitivity to a substance (allergen). Allergic symptoms include, but are not limited to, eczema, allergic rhinitis or nasal sensation, dry grass fever, conjunctivitis, bronchial asthma, urticaria (hives) and food allergies, and other atopic symptoms.

Allergies are generally caused by the production of IgE antibodies against harmless allergens. Cytokines induced by systemic or mucosal administration of immunomodulatory oligonucleotides are primarily in the class referred to as TH1 (eg, IL-12, IP-10, IFN-α and IFN-γ), which are these. Induces humoral and cell-mediated immune responses. Another major type of immune response associated with the production of IL-4 and IL-5 cytokines is referred to as the Th2 immune response. In general, allergic diseases appear to be mediated by a Th2-type immune response. Immune based on the ability of immunomodulatory oligonucleotides to shift the immune response in subjects from the major Th2 (related to the production of IgE antibodies and allergies) to the Th2 / Th1 balanced response (protective against allergic reactions). Effective dosages for inducing an immune response of a regulatory oligonucleotide can be administered to a subject to treat or prevent asthma and allergies.

Accordingly, the immunomodulatory oligonucleotide compositions provided have significant therapeutic utility in the treatment of allergic and non-allergic symptoms such as asthma. Th2 cytokines, especially IL-4 and IL-5, are elevated in the airways of asthmatic subjects. These cytokines promote key aspects of the asthmatic inflammatory response, including IgE isotope switching, eosinophil motivational factors, activation and mast cell proliferation. Th1 cytokines, especially IFN-γ and IL-12, can suppress the formation of Th2 clones and the production of Th2 cytokines. Asthma refers to a disorder of the respiratory system characterized by inflammation, narrowing of the airways, and increased responsiveness of the airways to inhalants. Asthma is often, but not limited to, associated with atopy or allergic symptoms.

The immunomodulatory oligonucleotide compositions provided may be administered in combination with antiallergic therapy. Conventional methods for treating or preventing allergies include the use of allergy medications or blunting therapies. Some evolutionary therapies for treating or preventing allergies include the use of neutralizing anti-IgE antibodies. Antihistamines and other drugs that interfere with the effects of chemical mediators of allergic reactions help control the severity of allergic symptoms, but do not prevent allergic reactions and have no effect on subsequent allergic responses. I don't have it. The blunting therapy is performed by giving a small amount of the allergen, usually by subcutaneous injection, to elicit an IgG-type response to the allergen. The presence of IgG antibodies is believed to help neutralize mediator production resulting from the induction of the IgE step. First, the subject is treated with a very low dose of the allergen so as not to elicit a severe response, then the dosage is slowly increased. This type of treatment is dangerous because the compound that causes the allergic response is actually administered to the subject and a severe allergic reaction can occur.

Anti-allergic agents include, but are not limited to, antihistamines, cortisteroids, and prostaglandin inducers. Antihistamines are compounds that neutralize histamine released by giant cells or basophils. These compounds are well known in the art and are commonly used in the treatment of allergies. Antihistamines include acrivastine, astemizole, azatazine, azelastine, betaastin, brompheniramine, buclyzine, cetirizine, cetirizine analog, chlorpheniramine, clematisne, CS 560, cyproheptadine, desloratazine, dexchlorphenylamine, evastin, epinastine. Includes, but is not limited to, xofenazine, HSR 609, hydroxyzine, levocabastine, loratidine, metoscoporamine, mizolastine, nolastemizine, phenindamine, promethazine, pyriramine, terphenazine, and tranilast. Corticosteroids include, but are not limited to, methylprednisolone, prednisolone, beclomethasone, budesonide, dexamethasone, flunisolide, flucicasone propionate, and triamcinolone. Dexametason is a corticosteroid with anti-inflammatory activity, but is constantly used for the treatment of allergies or asthma in inhaled form due to its high absorption and long-term inhibitory side effects at effective dosages. Not done. However, dexamethasone can be used in accordance with the present invention to treat allergies or asthma. This is because they can be administered at low doses to reduce side effects when administered in combination with the compositions of the invention. Side effects associated with corticosteroid use include coughing, speech disorders, and oral cataracts (candidiasis), and at higher doses systemic effects such as adrenal depression, glucose intolerance, osteoporosis, Includes aseptic necrosis of bones, cataract formation, growth inhibition, hypertension, muscle weakness, skin thinning, and easy bruising. Barnes and Peterson (1993) Am Rev Respir Dis 148: S1-S26; and Kamada AK et al. (1996) Am J Respir Crit Care Med 153: 1739-48.

The oligonucleotide compositions and methods provided according to the present invention can be used alone or in combination with other substances and methods useful for the treatment of asthma. In one aspect, the invention provides a method of treating a subject with asthma. A method according to this aspect of the invention comprises administering to a subject with asthma an effective amount of the composition of the invention to treat the subject.

In some embodiments, the invention provides a method of treating a subject having asthma. Methods according to aspects of the invention include the step of administering to a subject having asthma an effective amount of the composition of the invention and anti-asthma therapy for treating the subject.

As used herein, "asthma" refers to a respiratory disorder characterized by inflammation and narrowing of the airways, and increased responsiveness of the airways to inhalants. Asthma is often, but not limited to, associated with atopy or allergic symptoms. Symptoms of asthma include recurrent episodes of wheezing, shortness of breath, chest tightness and cough, resulting from airflow obstruction. Airway inflammation associated with asthma involves a number of physiological changes, including erosion of the airway mucosal epithelium, subbasement membrane collagen deposition, edema, giant cell activation, neutrophil, eosinophil and lymphocyte infiltration. Can be detected by observation of. As a result of airway inflammation, asthmatics usually experience airway hyperreactivity, airway restriction, respiratory symptoms, and chronic disease. Airflow restriction includes features that lead to acute acute bronchoconstriction, airway edema, mucus plug formation, and airway remodeling, generally bronchial obstruction. In some cases of asthma, subbasement membrane fibrosis may occur, which leads to persistent abnormalities in lung function.

Studies over the past few years have shown that asthma appears to result from complex interactions between inflammatory cells, mediators, and other cells and tissues present in the airways. Giant cells, eosinophils, epithelial cells, macrophages, and activated T cells all play important roles in asthma-related inflammatory processes. Djukanovic R et al. (1990) Am Rev Respir Dis 142: 434-457. It is believed that these cells can affect airway function through the secretion of preformed and newly synthesized mediators that can act directly or indirectly on local tissues. It has also been recognized that the T lymphocyte (Th2) subpopulation plays an important role in controlling allergic inflammation in the airways by releasing selective cytokines and establishing disease chronicity. Robinson DS et al. (1992) N Engl J Med 326: 298-304.

Asthma is a complex disorder that occurs at various stages of development and can be classified as acute, subacute or chronic based on the degree of symptoms. Acute inflammatory responses are associated with early recruitment of cells into the airways. The subacute inflammatory response involves cell recruitment and activation of resident cells, resulting in a persistent inflammatory pattern. Chronic inflammatory responses are characterized by persistent levels of cell damage and ongoing repair processes that can lead to permanent abnormalities in the airways.

A "subject with asthma" is a subject with a respiratory disorder characterized by inflammation and narrowing of the airways, and increased responsiveness of the airways to inhalants. Factors associated with the onset of asthma include, but are not limited to, allergens, cold temperatures, exercise, viral infections and SO ₂ .

As mentioned above, asthma may be associated with the Th2 cytokines IL-4 and IL-5, as well as the TH2 type of immune response, which is at least partially characterized by IgE antibody isomorphic substitutions. Th1 and Th2 immune responses are mutually antagonistic so that the tilt of the immune response to the Th1 type can suppress or alleviate the Th2 type of the immune response, including allergies. Therefore, the immunomodulatory oligonucleotide compositions provided by the present invention are useful in their own right for treating subjects with asthma. This is because analogs can tilt the immune response towards the Th1 type of immune response.

The immunomodulatory oligonucleotide compositions provided by the present invention may be administered in combination with asthma therapy. Conventional methods for treating or preventing asthma include antiallergic therapy (above) and the use of numerous other substances, including inhalants.

Medical care for the treatment of asthma generally falls into two categories: rapid palliative care and long-term management care. Asthma patients receive long-term care based on daily principles for achieving and maintaining persistent asthma management. Long-term care includes anti-inflammatory agents such as corticosteroids, sodium chromolin and nedocromil; long-acting bronchodilators such as long-acting β ₂ -agonists and methylxanthines; and leukotriene modifiers. Rapid palliative medicine includes short-acting β ₂ -agonists, anticholinergic drugs, and systemic corticosteroids. There are many side effects associated with each of these drugs, and none of the drugs, alone or in combination, can prevent or completely treat asthma.

Anti-asthma drugs include PDE-4 inhibitors, bronchial dilators / β-2 agonists, potassium (K ⁺ ) channel opening agents, VLA-4 antagonists, neurokinin antagonists, thrombin A2 (TXA2) synthesis inhibitors, xanthin, arachidone. It includes, but is not limited to, acid antagonists, 5 lipooxygenase inhibitors, TXA2 receptor antagonists, TXA2 antagonists, 5-lipox activating protein inhibitors, and protease inhibitors.

Bronchodilators / β ₂ agonists are a type of compound that causes bronchodilator or smooth muscle dilation. Bronchodilator / β ₂ agonists include, but are not limited to, salmeterol, salbutamol, albuterol, terbutaline, D2522 / formoterol, fenoterol, bitorterol, pyrubuerol methylxanthine, and orciprenaline. Long-acting β ₂ agonists and bronchodilators are compounds used for long-term symptom suppression in addition to anti-inflammatory therapy. Long-acting β ₂ agonists include, but are not limited to, salmeterol and albuterol. These compounds are usually used in combination with corticosteroids and are generally not used without any inflammatory therapy. They are associated with side effects such as tachycardia, skeletal muscle tremor, hypokalemia, and prolonged QTc intervals at overdose.

Methylxanthine contains, for example, theophylline and is used for long-term control and suppression of symptoms. These compounds cause bronchodilation, and possibly adenosine antagonism, resulting from phosphodiesterase inhibitors. Dose-related acute toxicity is a particular problem with these types of compounds. As a result, daily serum concentrations must be monitored to account for toxicity and narrow therapeutic range resulting from individual differences in metabolic clearance. Side effects include, but are not limited to, tachycardia, tachyarrhythmia, nausea and vomiting, central nervous system irritation, headache, seizures, hematemesis, hyperglycemia, and low potassium blood. Short-acting β ₂ agonists include, but are not limited to, albuterol, bitorterol, pirbuterol, and terbutaline. Some of the adverse effects associated with the administration of short-acting β ₂ agonists include tachycardia, skeletal muscle tremor, increased lactate, headache, and hyperglycemia.

Chromolysodium and nedocromil are used as long-term control medications primarily to control asthma symptoms resulting from exercise or allergic symptoms resulting from allergens. These compounds are believed to block early and delayed reactions to allergens by interfering with chloride channel function. They also stabilize the giant cell membrane and inhibit the activity and release of mediators from eosinophils and epithelial cells. Dosing for 4-6 weeks is generally required to achieve maximum benefit.

Anticholinergic drugs are commonly used to relieve acute bronchospasm. These compounds are thought to function by competitive inhibition of muscarinic choline receptors. Anticholinergic agents include, but are not limited to, ipratropium bromide. These compounds reverse only cholinergic-mediated bronchospasm and do not alter the response to the antigen. Side effects include dry mouth and respiratory secretions, increased wheezing in some individuals, and impaired vision when sprayed on the eyes.

In some embodiments, the immunomodulatory oligonucleotide compositions provided may be useful for treating airway remodeling. Airway remodeling results in smooth muscle cell proliferation and / or submucosal thickening in the airways, ultimately resulting in airway narrowing, which leads to restricted airflow. The immunomodulatory oligonucleotides of the present invention may suppress further remodeling and possibly even reduce the tissue build-up that results from the remodeling process.

In some embodiments, the immunomodulatory oligonucleotide compositions provided are useful for treating subjects with inflammatory disorders. As used herein, the term "inflammatory disorder" is associated with an antigen-nonspecific response of the innate immune system, including the accumulation and activation of leukocyte and plasma proteins, at the site of infection, toxin exposure or cell damage. Refers to symptoms. Cytokines characteristic of inflammation are tumor necrosis factor (TNF-α), interleukin 1 (IL-1), IL-6, IL-12, interferon α (IFN-α), and interferon β (IFN-β). , And chemokine. Thus, certain types of asthma, allergies, and autoimmune disorders may have the characteristics of inflammatory disorders. Inflammatory disorders also include, for example, cardiovascular disease, chronic obstructive pulmonary disease (COPD), bronchiectasis, chronic cholecystitis, tuberculosis, Hashimoto disease, septicemia, sarcoidosis, pneumoconiosis and other dust pneumonia, and transplantation in wounds. Including, but not limited to, foreign substances that have been treated. As used herein, the term "sepsis" refers to a well-known clinical syndrome associated with a host's systemic inflammatory response to microbial invasion. As used herein, the term "sepsis" refers to symptoms typically precursored by fever or hypothermia, tachycardia and rapid breathing, and in severe cases, hypotension, multiple organ failure, Even death can progress.

In some embodiments, the immunomodulatory oligonucleotide compositions provided are useful for treating subjects with an infectious disease. In some embodiments, the immunomodulatory oligonucleotide compositions provided are useful for treating an infectious disease vulnerable subject, including persons who may have been exposed to a pathogen or multiple pathogens. Immunomodulatory oligonucleotides used in accordance with the present invention may also be used in some embodiments to treat or suppress infections caused by viruses, bacteria, fungi, or parasites. A subject with an infectious disease is a subject who is exposed to an infectious pathogen and has an acute or chronic detectable level of the pathogen in the body. Immunomodulatory oligonucleotides can be used with or without antigen to enhance antigen-specific systemic or mucosal immune responses that can reduce the level of infectious pathogens or eradicate infectious pathogens. Infectious diseases as used herein are diseases that result from the presence of foreign microorganisms in the body. It is of particular importance to develop effective vaccine strategies and treatments to protect the mucosal surface of the body, which is the main site of pathogen invasion.

Viruses are small infectious agents that generally contain nucleic acid cores and protein coatings, but are not independent organisms. Viruses can also take the form of infectious nucleic acids lacking proteins. The virus cannot survive in the absence of cells that can replicate in it. The virus enters certain cells by either endocytosis or direct injection of DNA (phage) and proliferates to cause disease. The propagated virus can then be released and infect additional cells. Some of the viruses are DNA-containing viruses and others are RNA-containing viruses. DNA viruses include pox, herpes, adeno, papova, parvo, and hepadona hepadona. RNA viruses include picorna, calici, astro, toga, flavi, corona, paramixo, orthomixo, bunya, arena, rabdo, fio, borna, leo and retro. In some embodiments, the invention also presents a disease in which prions are associated with disease progression, such as bovine spongiform encephalopathy (ie, mad cow disease, BSE) or animal scrapie infection, or human Creutzfeldt-Jakob disease. Intended to treat.

Viruses include enteroviruses (including but not limited to viruses of the Picornaviaceae family, such as poliovirus, Coxsackie virus, echovirus), rotavirus, adenovirus, and hepatitis virus, such as A, B, C. , But not limited to hepatitis D and E viruses. Specific examples of viruses found in humans include, but are not limited to: Retroviridae (eg, human immunodeficiency virus, eg HIV-1 (HTLV-III, LAV or HTLV-). III / LAV, also referred to as HIV-III); and other isolates, such as HIV-LP; Picornaviridae (eg, poliovirus, hepatitis A virus; enterovirus, Coxsackie). Virus, renovirus, echovirus); Calciviridae (eg, strain that causes gastroenteritis); Togaviridae (eg, horse encephalitis virus, rubellavirus); Flaviviridae (Flaviviridae) ( For example, dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (eg, coronavirus); Rhabdoviridae (eg, bullous stomatitis virus, mad dog disease virus); Filoviridae (Ebola virus, for example); Paramyxoviridae (eg, parainfluenza virus, mumps virus, hemp virus, respiratory rash virus); Orthomyxoviridae (eg, influenza virus); Bunyavirus family (eg, influenza virus) Bunyaviridae) (eg Hantan virus, Bunya virus, Frevo virus, and Nairo virus); Arenaviridae (hemorrhagic fever virus); Reoviridae (eg Leovirus, Orbi) Virus and Rotavirus); Birnaviridae; Hepadnaviridae (Hepatinavirus B virus); Parvoviridae (Parvoviridae); Papovavihdae (Papovavihdae) (Papillomavirus, poly) Omavirus); Adenoviridae (mostly adenovirus); Herpesviridae (Herpesviridae) (Herpes simple virus (HSV) types 1 and 2, varicella / band Herpes virus, cytomegalovirus (CMV); Bronchitis virus, Alphavirus, Kaposi sarcoma-related herpes virus, Newcastle disease virus, Nipah virus, Norwalk virus, Papillomavirus, Parainfluenza virus, Bird influenza, SAR virus, West Nile fever. Viruses that infect plants include, for example, viruses of the following families and / or genera: Alphaviruses: Bromoviridae, Alphacryptoviruses: Partitiviridae, Badnaviruses (Partitiviridae). Badnaviruse), Betacryptoviruses: Partitiviridae, Bigeminiviruses: Geminiviridae, Bromoviruses: Bromoviridae, Bymoviruses: Pochi Viralidae (Potyviridae), Capilloviruses; Carlaviruses, Carmovirus virus: Tombusviridae, Caulimoviruses, Closteroviruses, Comovirus Viruses (Comovirus viruses): Comoviridae, Cloosteroviruses, Comoviruses: Comoviridae, Cucumoviruses: Bromoviridae , Cytorhabdoviruses: Rhabdoviridae, Dianthoviruses, Enamoviruses, Fabaviruses: Comoviridae, Fijiviruses: Leo Viralidae (Reoviridae), Furoviruses, Hordeiviruses, Hybrigeminiviruses: Geminiviridae, Idaeovirus iruses), Ilarviruses: Bromoviridae, Ipomoviruses: Potyviridae, Luteoviruses, Machlomoviruses, Macluraviruses, Mala Marafiviruses, Monogeminiviruses: Geminiviridae, Nanaviruses, Necroviruses, Nepoviruses: Comoviridae, Nucleo Nucleorhabdoviruses: Rhabdoviridae, Oryzaviruses: Reoviridae, Ourmiaviruses, Phytoreoviruses: Reoviridae, Potexvirus Potexviruses), Potyviruses: Potyviridae, Rymoviruses: Potyviridae, Satellite RNA, Satelliteviruses, Sequiviruses: Sequiviridae , Sobemoviruses, Tenuiviruses, Tobamoviruses, Tobraviruses, Tombusviruses: Tospoviruses: Bunyaviridae, Trichoviruses, Chimovirus (Tymoviruses), Umbraviruses, unattributable Pochivirus: Potyviridae, unattributable Labdovirus family: Varicosaviruses, Waikavirus (Waikaviruses): Sequiviridae, unclassified virus, bromovirus, potivirus and timovirus; certain related plant viruses are, for example, Tobacco mosaic virus (TMV), tomato spotted virus. Tomato spotted wilt virus, Tomato yellow leaf curl virus, Cucumber mosaic virus, Potato virus Y, Cauliflower mosaic virus, Africa African cassava mosaic virus, Plum pox virus, Brome mosaic virus, Potato virus X, Citrus tristeza virus, Omugi Barley yellow dwarf virus, Potato leafroll virus, and Tomato bushy stunt virus.

Viral hepatitis is an inflammation of the viscera that can cause swelling, tenderness, and sometimes permanent damage to the liver. If the liver inflammation persists for at least 6 months, it is called chronic hepatitis. There are at least five viruses known to cause viral hepatitis, including hepatitis A, B, C, D and E. Hepatitis A is commonly transmitted by food or drinking water contaminated with human feces. Hepatitis B generally spreads through body fluids such as blood. For example, it can spread from the mother to the child at birth via sexual intercourse, contaminated blood infusions and needles. Hepatitis C is quite common, similar to hepatitis B, and is commonly prevalent via blood infestations and contaminated needles. Hepatitis D is most often found in IV drug users who carry the hepatitis B virus with which it is associated. Hepatitis E is similar to viral hepatitis A and is generally associated with poor hygiene.

Gram-negative and Gram-positive bacteria act as antigens in vertebrates. Such Gram-positive bacteria include, but are not limited to, the genera Pasteυrella, Staphylococci, and Streptococcus. Gram-negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas, and Salmonella. Specific examples of infectious bacteria include Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, and the genus Mycobacteria (eg, M. chu). Belcrosis, M. Avium, M. Intracellulare, M. Cansassie, M. Gordnae, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis , Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (Group B Streptococcus) Streptococcus faecalis), Streptococcus bovis, Streptococcus (anaerobic genus), Streptococcus
Streptococcus pneumoniae, pathogenic Campylobacter, Enterococcus, Haemophilus influenzae, Bacillus antracis, Corynebacterium diphtheria Corynebacterium, Erysipelothrix rhusiopathiae, Clostridium pen'ringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella leneumonee Pasturella multocida, Bacteroides, Fusobacterium nucleatum,

Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces israeli (Actinomyces) , Not limited to these.

Examples of fungi are Cryptococcυs neoformans, Histoplasma capsulatum, Cocccidioides immitis, Blastomyces dermatitis, tratomyces dermatitidis, and Blastomyces dermatitidis. Includes Candida albicans.

Other infectious organisms (ie, protozoa) include the genus Plasmodium, such as Plasmodium falciparum, Plasmodium malariae, and Plasmodium ovale (Plasmodium malariae). Includes Plasmodium ovale), Plasmodium vivax, and Toxoplasma gondii.
Blood-borne parasites and / or tissue parasites include the genus Plasmodium, Babesia microti, Babesia divergens, Leishmania tropica, and Leishmania. , Leishmania braziliensis, Leishmania donovani, Trypanosoma gambiense and Trypanosoma rhodesiense (African sleep disease), Trypanosoma cru (African sleep disease) Leishmania), and includes Toxoplasma gondii.

Other pharmaceutically relevant microorganisms are extensively described in the literature, see, for example, CGA Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983, the entire contents of which are incorporated herein by reference. .. Agriculturally related microorganisms (eg, microorganisms that infect crops and / or livestock) are also known, such as George N. Agrios, Plant Pathology, Elsevier Academic Press, 5th Edition, 2005; John Lucas, Plant Pathology and Plant Pathogens, Wiley-Blackwell; 3rd Edition, 1998; Dwight C. Hirsh, N. James MacLachlan, Richard L. Walker, Veterinary Microbiology, Wiley-Blackwell; 2 edition, 2004; and PJ Quinn, et al, Veterinary Microbiology and Microbial Disease, Wiley-Blackwell; includes, but is not limited to, the microorganisms described in 2nd Edition, 2011.

The oligonucleotide compositions provided by the present invention may be administered to a subject (human subject, or in some embodiments, an animal (eg, livestock or pet) or plant (eg, crop subject)) with an antibacterial agent. Antibacterial agents used herein refer to natural or synthetic compounds capable of killing or inhibiting infectious microorganisms. Types of antibacterial agents useful in accordance with the present invention are those infecting or infecting a subject. It will depend on the type of microorganism at risk. Antibacterial agents include, but are not limited to, antibacterial agents, antiviral agents, antifungal agents and antiparasitic agents. "Anti-infective agents", "antibacterial agents" , "Antiviral agents", "Antibacterial agents and", "Antiparasitic agents" and "Antibacterial agents" have familiar meanings to those skilled in the art and are standard medical texts. That is, an antibacterial agent kills or inhibits a bacterium and contains an antibiotic and other synthetic or natural compounds having similar functions. An antibiotic is a low molecular weight produced as a secondary metabolism by a cell. Molecules such as microorganisms. In general, antibiotics interfere with one or more bacterial functions or structures that are specific to the microorganism and are not present in the host cell. Antiviral agents are from natural sources. It can be isolated or synthesized and is useful for killing or inhibiting the virus. Antibacterial agents are used to treat superficial fungal infections as well as opportunistic and major systemic fungal infections. Used. Antibacterial agents kill or inhibit parasites.

Examples of antiparasitic agents, also referred to as parasites useful for human administration, are alvendazole, amphotericin B, benznidazole, bitionol, chloroquine HCl, chloroquine phosphate, clindamycin, dehydroemetine, diethylcarbamazine. , Fronate diloxanide, eflornitin, frazoridaone, glucocorticoid, halophanthrin, iodoquinol, ibermectin, mebendazole, meflokin, antimegrumione, meralsoprol, methrihonate, metronidazole, niclosamide, niflutimox, oxamnikin, paromomycin Pentamidin, piperazin, prazicantel, primaquine phosphate, proguanyl, pyranthel pamoate, pyrimethanmin sulfonamide, pyrimethanmin sulfadoxin, quinacrine HCl, quinine sulfate, quinidine gluconate, spiramycin, sodium stivogluconate (antimonate gluconate) Includes, but is not limited to, sodium), slamin, tetracycline, doxicycline, thiabendazole, quinidazole, trimetprim-sulfametozazole, and tripulsamide. Some of them are used alone or in combination with others.

Antibacterial agents kill or inhibit the growth or function of bacteria. A large class of antibacterial agents are antibiotics. Antibiotics that are effective in killing or inhibiting a wide range of bacteria are referred to as broad-spectrum antibiotics. Other types of antibiotics are primarily effective against Gram-positive or Gram-negative classes of bacteria. These types of antibiotics are referred to as narrow spectrum antibiotics. Other antibiotics that are effective against a single organism or disease but not against other types of bacteria are referred to as restricted spectrum antibiotics. Antibacterial agents may be classified based on their predominant mode of action. In general, antibacterial agents are cell wall synthesis inhibitors, cell membrane inhibitors, protein synthesis inhibitors, nucleic acid synthesis or functional inhibitors, and competitive inhibitors.

An antiviral agent is a compound that suppresses viral infection of a cell or replication of the virus in the cell. There are far fewer antivirals than antibacterial drugs. Because the process of viral replication is so closely associated with the replication of DNA in the host cell, non-specific antiviral agents are generally toxic to the host. There are several stages in the process of viral infection that can be blocked or inhibited by antiviral agents. These steps include viral adhesion to host cells (immunoglobulin or binding peptide), viral decoating (eg, amantazine), synthesis or translation of viral mRNA (eg, interferonron), viral RNA or Includes RNA replication (eg, nucleotide analogs), maturation of novel viral proteins (eg, protease inhibitors), and viral germination and release.

Nucleotide analogs are synthetic compounds that resemble nucleotides but have incomplete or abnormal deoxyribose or ribose groups. Once the nucleotide analogs are intracellular, they are phosphorylated to produce a formed triphosphate ester that antagonizes normal nucleotides for uptake into viral DNA or RNA. Once the nucleotide analog triphosphate form is incorporated into the growing nucleic acid chain, it causes irreversible association with viral polymerases, thus causing chain termination. Nucleic acid analogs are acyclovir (used for the treatment of herpes simple virus and varicella-zoster), ganciclovir (useful for the treatment of cytomegalovirus), idoxuridine, ribavirin (respiratory polynuclear virus). (Useful for the treatment of), dideoxyinosin, dideoxycitidine, zidovudine (azidothymidin), imikimod, and reshikimod, but not limited to these.

Interferon is a cytokine secreted by virus-infected cells and immune cells. Interferon functions by binding to specific receptors on cells adjacent to infected cells, causing changes in the cells that protect the cells from infection by the virus; α and β-interferons are also on the surface of infected cells. Induces the expression of class I and class II MHC molecules, resulting in increased antigen presentation for host immune cell recognition; α and β-interferon are available as recombinants and of chronic hepatitis B and C infections. Used for treatment. At dosages that are effective for antiviral therapy, interferon has severe side effects such as fever, upset and weight loss.

Antiviral agents useful in the present invention include, but are not limited to, immunoglobulins, amantadine, interferons, nucleotide analogs, and protease inhibitors. Specific examples of antiviral agents include acyclovir; acyclovir sodium; adefovir; allodin; alvircept Sudotox; amantadine hydrochloride; alanothin; allyldon;
Atevirdin mesylate; Ablysine; Sidfovir; Cypamphyllin; Citalabine hydrochloride; Delavirdine mesylate; Descyclovir; Didanosine; Disoxaril; Edxdin; Enviraden; Enviroxime; Famichiclovir; Famotin hydrochloride; Fiasitabin; Fiallysine; Foscarnet sodium; Sodium; ganciclovir; ganciclovir sodium; idoxuridine; ketoxal; ramibdin; lobukavir; memotin hydrochloride;
Methisazone; nevirapine; pencyclovir; pyrodavir; ribavirin; limantazine hydrochloride; saquinavir mesylate; somantazine hydrochloride; sorivudine; statron; stubzin; tyrolone hydrochloride; trifluridine; baracyclovir hydrochloride; vidarabine; vidarabine phosphate; vidarabine sodium phosphate; And, but not limited to, zimbiloxime.

Antifungal agents are useful for the treatment and prevention of infectious fungi. Antifungal agents are sometimes classified by their mechanism of action. Some antifungal agents act as cell wall inhibitors by inhibiting glucose synthase. They are,

Includes, but is not limited to, Bashiungin / ECB. Other antifungal agents work by destabilizing membrane integrity. These include imidazoles such as clotrimazole, sertaconazole, fluconazole, intraconazole, ketoconazole, miconazole and voriconazole, as well as FK 463, amphotericin B, BAY 38-9502, MK 991, pradimycin, UK 292, butenafine, And, but not limited to, terbinafine. Other antifungal agents function by cleaving chitin (eg, chitinase) or by immunosuppression (501 cream).

In some embodiments, the immunomodulatory oligonucleotide compositions provided are useful for treating subjects with cell proliferation disorders such as cancer. A subject with cancer is a subject with detectable cancerous cells. The cancer can be malignant or non-malignant. Cancers or tumors are bile duct cancer; brain tumor; breast cancer; cervical cancer; villous cancer; colon cancer; endometrial cancer spiral; esophageal cancer; gastric cancer; intraepithelial tumor; lymphoma; liver cancer; lung cancer (eg, small cells and non-cancer) Small cells); melanoma; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; rectal cancer; sarcoma; skin cancer; testicle cancer; thyroid cancer; and kidney cancer, and other malignant tumors and sarcoma. , Not limited to these. In some embodiments, the cancer is hairy cell leukemia, chronic myeloid leukemia, cancerous T cell leukemia, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, kidney cancer, prostate cancer, bladder cell carcinoma. , Or colon cancer.

The immunomodulatory oligonucleotide compositions provided can be administered alone or in combination with anti-cancer therapy. Anticancer therapies include, but are not limited to, radiation therapy, chemotherapy, immunotherapy, or cancer vaccines, hormonal therapies, biological response modifiers, and surgical means. A cancer drug means a substance that is administered to a subject for the purpose of treating cancer. As used herein, "treating cancer" includes preventing the progression of cancer, reducing the symptoms of cancer, and / or inhibiting the growth of established cancer. In another aspect, the cancer drug is administered to a subject at risk of developing cancer with the aim of reducing the risk of developing cancer. Various types of pharmaceuticals for the treatment of cancer are described herein. For this particular purpose, cancer drugs are classified as chemotherapeutic agents, immunotherapeutic agents, cancer vaccines, hormonal therapies and biological response modifiers.

Furthermore, the methods of the invention are intended to include the use of more than one pharmaceutical with the immunomodulatory oligonucleotide compositions provided. By way of example, preferably, the provided immunomodulatory oligonucleotide composition can be administered with both a chemotherapeutic agent and an immunotherapeutic agent. Alternatively, the cancer drug may include immunotherapeutic agents and cancer vaccines, or chemotherapeutic agents and cancer vaccines, or chemotherapeutic agents, immunotherapeutic agents and cancer vaccines, all of which have or are at risk of developing cancer. It is administered to one subject for the purpose of treating the subject in.

Chemotherapeutic agents can be selected from the group consisting of, but not limited to: methotrexate, vincristin, adriamycin, cisplatin, non-sugar-containing chloroethylnitrosourea, 5-fluorouracil, mitomachine C, bleomycin, doxorubicin, Doxorubicin, taxol, fragiricin, megramin GLA, valrubicin, carmstein and polyferposan, MMI270, BAY 12-9566, RAS famesyl transferase inhibitor, famesyl transferase inhibitor, MMP, MTA / LY231514, LY264618 / Rometexol, Gramorec, CI-994, TNP-470, High Kamchin / Topotecan, PKC412, Balspodal / PSC833, Novantron / Mitoxanthron, Metallet / Slamin, Bachimasut, E7070, BCH-4556, CS- 682, 9-AC, AG3340, AG3433, Insel / VX-710, VX-853, ZD0101, ISI641, ODN 698, TA 2516 / Marumistat, BB2516 / Marumistat, CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202 , FK 317, pisibanil / OK-432, AD 32 / valrubicin, metastron / strontium derivative, temodar / temozoromid, evaset / liposomal doxorubicin, yeutaxel / paclitaxel, taxol / paclitaxel, Xeloda / capecitabin, fluturon / doxyllysine Paclitaxel, Oral Toxoid, SPU-077 / Sisplatin, HMR 1275 / Flabopyridol, CP-358 (774) / EGFR, CP-609 (754) / RAS Cancer Gene Inhibitor, BMS-18751 / Oral Platinum, UFT (Tegaful) / Urasyl), ergamizol / levamisol, enylrasyl / 776C85 / 5FU enhancer, campto / levamizol, camptosal / irinotecan, tomudex / lartitrexed, leustatin / cladribin, paclitaxel / paclitaxel, doxil / liposomeized doxorubicin, Ludala / Fludarabine, Falmorubicin / Epirubicin, Depot Cyt, ZD1839, LU 79553 / Bis-Naphtalimide, LU 103793 / Drastatin, Kaetix / Liposomal doxorubicin, Gemzar / Gemcitabine, ZD 0473 / Anormed, YM 116 , CDK4 and CDK2 inhibitors, PARP inhibitors, D4809 / dexfosamide, ifes / mesnex / ifosamide, bumon /
Teniposide, paraplatin / carboplatin, plantinol / carboplatin, plantinol / cisplatin, bepeside / etoposide, ZD 9331, taxotere / docetaxel, guanine arabinoside prodrugs, taxane analogs, nitrosourea, alkylating agents such as melferan And cyclophosphamide, aminoglutetimide, asparaginase, busulfan, carboplatin, chlorambusyl, citarabin HCl, taxinomycin, daunorbisin HCl, estramstine sodium phosphate, etoposide (VP16-213), fluoroxylidine, fluorouracil (5) -FU), flutamide, hydroxyurea (hydroxyurea), isosphamide, interferon α-2a, α-2b, leuprolide acetate (LHRH-releasing factor analog), lomustine (CCNU), meiloletamine HCl (nitrogen mustard), mercaptopurine, mesna , Mitotan ( ^op' -DDD), Mitoxantron HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozosine,

Tamoxiphene citrate, thioguanine, thiotepa, vinblastine sulfate, amsacrine (m-AMSA), azacitidine, erythropoietin, hexamethylmelamine (HMM), interleukin 2, mitoguazone (methyl-GAG; methylglioquilvis-guanylhydrazone) MGBG), pentostatin (2'-deoxycoformycin), semustine (methyl-CCNU), teniposide (VM-26) and vinblastine sulfate.

Immunotherapeutic agents include Ributaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolim, SMART M195, Atlagen, Ovalex, Bexal, LDP-03, ior tβ, MDX-210, MDX-11, MDX -22, OV103, 3622W94, Anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, Sheaside, Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000, Group consisting of LymphoCide, CMA 676, monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART 1 D10 Ab, SMART ABL 364 Ab, and ImmuRAIT-CEA Can be selected, but is not limited to these.

Cancer vaccines include EGF, anti-iodoid cancer vaccine, Gp75 antigen, GMK melanoma vaccine, MGV ganglioside complex vaccine, Her2 / neu, Ovarex, M-Vax, O-Vax, L-Vax, STn-KHL theratope, BLP25 ( A group consisting of MUC-1), liposomal iodine vaccine, melatin, peptide antigen vaccine, toxin / antigen vaccine, MVA-type vaccine, PACIS, BCG vaccine, TA-HPV, TA-CIN, DISC-virus and ImmuCyst / TheraCys. You can choose from, but you are not limited to them.

As used herein, the terms "cancer antigen" and "tumor antigen" are compatible to refer to antigens that are differentially expressed by cancer cells and thereby can be utilized to target cancer cells. Used for Cancer antigens are clearly antigens that can potentially stimulate a tumor-specific immune response. Some of these antigens are not necessarily expressed by normal cells, but are encoded. These antigens are characterized as those that are normally silent (ie, not expressed) in normal cells, those that are expressed only at specific stages of differentiation, and those that are transiently expressed, such as embryonic and fetal antigens. Attached. Other cancer antigens are encoded by mutant cell genes such as cancer genes (eg activated ras cancer genes), suppressor genes (eg mutant p53), fusion proteins obtained from internal deletion or chromosomal translocation. .. Yet yet other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA oncoviruses.

In some embodiments, the immunomodulatory oligonucleotide compositions provided may also be useful for treating and preventing autoimmune diseases. Autoimmune diseases are diseases in which the subject's own antibody reacts with the host tissue or the immune effector T cells are self-reactive to the endogenous autopeptide and cause tissue collapse. Therefore, an immune response is made against the subject's own antigen, called the self-antigen. Autoimmune diseases include round alopecia, acquired hemophilia, tonic cough, antiphospholipid antibody syndrome, autoimmune-related infertility, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune hemolytic anemia. , Autoimmune diabetes, autoimmune thrombocytopenic purpura, Bechet's disease, vesicular vesicles, myocardial disease, chronic fatigue immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuritis, Churg-Strauss syndrome , Ocular scarring tinea, CREST syndrome, cold agglutinosis, dermatomyitis, discoid erythema, essential mixed cryoglobulinemia, fibromyalgia, fibromyitis, Gillan Valley syndrome, Hashimoto Diseases, glomerulonephritis (eg, rapidly progressive glomerulonephritis, proliferative glomerulonephritis), Graves' disease, transplant-to-host disease, Good Pasture syndrome, vesicles (eg, vulgaris vulgaris), idiopathic lung fibers Disease, idiopathic thrombocytopenic purpura, insulin resistance, idiopathic azison disease, IgA nephropathy, inflammatory bowel disease (including Coulomb disease and ulcerative colitis), juvenile arteritis, squamous lichenitis, severe muscle Asthenia, polysclerosis, mixed connective tissue disease, polymyositis, malignant anemia, nodular polyarteritis, polychondritis, polyglandular syndrome, rheumatic polymyopathy, primary agammaglobulinemia , Primary biliary cirrhosis, psoriasis, Lehnor's phenomenon, Reiter's syndrome, juvenile and adult rheumatoid arthritis, Schegren's syndrome, anti-collagen antibody-related scleroderma, Stiffperson's syndrome, systemic erythema (SLE), hyperan arteritis, Includes, but is not limited to, organ transplant refusal, temporal arteritis / giant cell arteritis, vaginitis, ulcerative colitis, vasculitis, and leukoplakia vulgaris.

As used herein, "self-antigen" refers to an antigen in a normal host tissue. Normal host tissue does not contain cancer cells. Thus, in the context of self-immunity, an immune response that occurs against a self-antigen is an unwanted immune response that causes tissue collapse and damage, whereas an immune response that occurs against a cancer antigen is a desirable immune response. It is a response and contributes to the disintegration of the tumor or cancer. Therefore, in some embodiments of the invention aimed at treating an autoimmune disease, it is not recommended that the immunomodulatory oligonucleotide be administered with an autoantigen, particularly an autoantigen that is the target of the autoimmune disease. ..

In another example, the immunomodulatory oligonucleotide used according to the invention can be delivered with a dosage of self-antigen. Numerous animal studies have demonstrated that mucosal administration of low-dose antigens results in immunocompromised or "tolerant" conditions. The active mechanism appears to be cytokine-mediated immune induction (ie, TGF-β control) away from Th1 primarily against Th2 and Th3. Active suppression by low-dose antigens also suppresses unrelated immune responses that are of great interest in the treatment of autoimmune diseases such as rheumatoid arthritis and SLE (bystander effect). Binder effects include the secretion of Th1 antagonistic regulatory, inhibitory cytokines in a local environment where pre-inflammatory and Th1 cytokines are released either in an antigen-specific or non-antigen-specific manner. As used herein, "tolerance" refers to this phenomenon. In fact, oral tolerance is a large number of autoimmune diseases in animals, including experimental autoimmune encephalomyelitis (EAE), experimental autoimmune myasthenia gravis, collagen-induced arthritis (CIA), and insulin-dependent diabetes mellitus. It has become effective in the treatment of. In these models, prevention and suppression of autoimmune diseases is associated with a shift in antigen-specific humoral and cellular responses from Th1 to Th2 / Th3 responses.

Non-limiting examples of useful immunomodulatory oligonucleotides that can be used in accordance with the present invention are provided in Attachment (B).

RIPtide
In some embodiments, the oligonucleotide utilized in accordance with the present invention is, or acts as an RNA-interacting polynucleotide (“RIPtide”). RIPtide is one of the therapies intended to open up the void potential of "undruggable" targets by inactivating the structural RNA associated with the disease. RIPtide is typically a small nucleotide sequence (about 8 nucleotides) that binds and divides the action of structural RNA. Due to their size, these molecules can easily cross the cell membrane. See, for example, US Pat. No. 6,080,585.

RIPtide can be useful for several clinical applications. In the context of cancer treatment, at least one target is telomerase, which is present in about 90% of cancer cells. Thus, in some embodiments, the sterically controlled oligonucleotides of the invention can be used to target telomerase in a variety of cancer therapeutic applications. In addition, RIPtide may be useful for infectious disease treatment applications. Viruses such as HIV and hepatitis C need to be able to hijack host cells in order to replicate. Therefore, structural RNA can play an important role in the maturation and replication of many viruses. Thus, in some embodiments, the sterically controlled oligonucleotides of the invention can be used to target a portion of DNA or RNA involved in viral maturation and / or replication.

Therefore, the present invention is useful for treating various viral infectious diseases. Viruses include, but are not limited to, enteroviruses (including but not limited to picornaviruses such as poliovirus, coxsackievirus, echovirus), rotavirus, adenovirus, and types A, B, C, D and E. Examples include, but are not limited to, hepatitis viruses such as hepatitis. Specific examples of viruses found in humans include: Retroviridae (eg, human immunodeficiency viruses such as HIV type 1, (also referred to as HTLV-III), LAV or HTLV-III / LAV, or HIV-III; and HIV. -Other isolates such as LP; Picornavirus family (eg, poliovirus, hepatitis A virus; enterovirus, human coxsackie virus, rhinovirus, echovirus); calicivirus family (eg, strains responsible for gastroenteritis) Togavirus family (eg, horse encephalitis virus, ruin virus); Flavivirus family (eg, dengue virus, encephalitis virus, yellow fever virus); Coronavirus family (eg, coronavirus); Rabdovirus family (eg, bullous stomatitis) Virus, mad dog disease virus); Phyllovirus family (eg, Ebola virus); Paramixovirus family (eg, parainfluenza virus, mumps virus, measles virus, RS virus); Orthomixovirus family (eg, influenza virus); Bunyavirus family (Eg, Hantan virus, Bunia virus, Frevovirus and Nyrovirus); Arenaviridae (hemorrhagic fever virus); Leovirus family (eg, Leovirus, Orbivirus and Rotavirus); Burnavirus family; Hepadnavirus Family (Hepatitis B virus); Parvovirus family (Parvovirus); Papovavirus family (papillomavirus, polyomavirus); Adenovirus family (mainly adenovirus); Herpesvirus family (simple herpesvirus (HSV) type 1 and Type 2, varicella zoster virus, cytomegalovirus (CMV)); Poxvirus family (squash virus, vaccinia virus, Poxvirus); Iridovirus family (eg, African pig cholera virus); and other viruses acute laryngeal bronchi Examples include, but are not limited to, flame virus, alpha virus, Kaposi sarcoma-related herpes virus, Newcastle disease virus, niper virus, nowalk virus, papillomavirus, parainfluenza virus, triinfluenza, SARS virus, and Westnile virus.

Antisense Drugs The present invention also includes various antisense-based therapies. Typically, the antisense drug is a small DNA-like or RNA-like compound (12-21 nucleotides) chemically modified to design favorable drug properties. Some antisense agents useful in the art include phosphorothioates or deoxyoligonucleotides, in which the sulfur group is exchanged for non-crosslinked oxygen on the phosphate skeleton. This increases resistance to ribonuclease degradation and improves pharmacological stability. In addition, in other agents, the so-called "next generation antisense molecules," the oligonucleotide is a 2'-O-methoxyethyl modification of the backbone, which increases the affinity of the target RNA for these oligonucleotides. I can let you. However, in some cases, many of these agents have side effects such as unwanted immune responses. In at least some situations, the undesired properties of such agents are due, at least in part, to their stereorandom nature. The present invention provides sterically controlled equivalents with favorable properties such as improved lipophilicity and increased RNA binding.

Several antisense-based therapies have been developed or are under development. The following are unrestricted examples of antisense therapy. The sterically controlled (eg, chiral pure) oligonucleotides described herein have improved efficacy, increased affinity for targets, and side effects compared to antisense drugs currently available in the art. Can be synthesized to achieve reduced pharmacokinetics, enhanced stability and / or increased bioavailability. In some embodiments, the antisense therapy includes morpholino drugs. For example: Morcos, PA (2007). "Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos". See Biochem Biophys Res Commun 358 (2): 521-7.

Cytomegalovirus Retinitis: Fomivirsen (commercially available as Vitraben) was approved by the US Food and Drug Administration in August 1988 for the treatment of cytomegalovirus retinitis.

Hemorrhagic Fever Virus: In early 2006, scientists at the U.S. Army Medical Research Institute for Ebola hemorrhagic fever virus research infect four red lizards with an antisense morpholino drug developed by the U.S. biotechnology company Abibiopharma. After treatment, he announced that he had recovered 75%. [US Army Medical Research Institute, Fort Detrick, Maryland. Newspaper announcement: Gene-specific Ebola treatment protects non-human primates from fatal diseases. January 13, 2006] The normal mortality rate for monkeys infected with the Ebola virus is 100%. In late 2008, Abibiopharma successfully filed an investigational drug (IND) application with the US Food and Drug Administration for its two lead products for the Marburg virus and Ebola virus. These drugs, AVI-6002 (Lu, X; Yu, Q; Binder, GK; Chen, Z; Slepushkina, T; Rossi, J; Dropulic, B (2004). "Antisense-Mediated Inhibition of Human Immunodeficiency Virus (HIV) ) Replication by Use of an HIV Type 1-Based Vector Results in Severely Attenuated Mutants Incapable of Developing Resistance ". A novel analog based on the PMO antisense chemical of AVI, enhanced by the addition of the positively charged component of. Preclinical results for AVI-6002 and AVI-6003 showed reproducible high survival rates for non-human primates exposed to the lethal infections of Ebola and Marburg viruses, respectively (Medical News Today. Abibio). Pharma announced that the US Food and Drug Administration will approve an investigational drug application for clinical trials of RNA therapeutics for the treatment of Ebola and Marburg virus (December 30, 2008).

Cancer: Also in 2006, German physicians reported a dose of compound AP12009 (phosphorothioate antisense oligodeoxynucleotide specific for the mRNA of human transforming growth factor TGF-β2) in patients suffering from high-grade glioma. Reported an increasing study. At the time of reporting, no median overall survival was obtained, and the authors suggested a potential therapeutic agent (as a result of G004, compound AP12009 targeting TGF-b2 in recurrent undifferentiated astrocytomas. Phase IIb Active Control Clinical Trials-Hau et al. 24 (18 Supplement): 1566-Abstracts of the American Society of Clinical Oncology Meetings).

For example, Trabedersen (AP12009-P001) is being developed as a monotherapy for the treatment of patients suffering from advanced pancreatic cancer, malignant melanoma, brain tumors, undifferentiated astrocytomas, and colorectal cancer. AP12009-P001 is given intravenously to 61 patients with advanced solid tumors known to overproduce TGF-b2, who are no longer treatable or no longer treatable with established therapies. Is being evaluated in an open-label, multicenter, Phase I / II dose escalation study to evaluate the safety and tolerability of the drug.

Other examples of antisense agents targeting proteins overexpressed in cancer are: clusterlin (OGX), which regulates epithelial interlobular conversion induced by growth factor TGFβ and transcriptional regulator TWIST1 in prostate cancer cells. -111 / TV01011); ATL1101 for the treatment of prostate cancer (next-generation antisense inhibitor of insulin-like growth factor I receptor (IGF-IR)); oblimersen for the treatment of chronic lymphocytic leukemia (trade name: Genasense); chronic bone marrow G4460 and LR3001 (c-myb inhibitor) for the treatment of sex leukemia, melanoma, neuroblastoma, and breast, pancreas and colon cancer; MG98 (DNA methyltransferase I inhibitor) for the treatment of renal cell carcinoma; ISIS5132 ( c-Raf antisense); LY90003 (protein kinase Cα antisense); G3139 (Bcl-2 antisense), and the like.

HIV / AIDS: Beginning in 2004, US researchers are conducting research using antisense technology to combat HIV. It is known that antisense-mediated inhibition of human immunodeficiency virus (HIV) replication by the use of the HIV type 1 base vector results in a highly attenuated variant in which resistance cannot be developed. In February 2010, researchers recovered the patient T, modified it to the HIV viral envelope protein using RNA antisense strand, and reinjected into the patient during the planned course of retroviral drug therapy. We reported that we succeeded in reducing the amount of HIV virus using cells.

High Cholesterol: In 2010, Mipomersen (formerly ISIS301012, trade name Kynamro) successfully completed Phase III trials for several types of high cholesterol. Mipomersen is a candidate for cholesterol-lowering drugs. It is an antisense therapy targeting messenger RNA against apolipoprotein B. See Merki E, Graham MJ, Mullick AE, et al. (August 2008). "Antisense oligonucleotides targeting human apolipoprotein B-100 reduce the amount of lipoprotein (a) and oxidized phospholipids on human apolipoprotein B-100 particles in lipoprotein (a) transgenic mice." .. Circulation 118 (7): 743-53; El Harchaoui K, Akdim F, Stroes ES, Trip MD, Kastelein JJ (2008). "Low-density lipoprotein-current and future pharmacological options for the management of patients who cannot achieve cholesterol are achieved with statins." Am J Cardiovasc Drugs 8 (4): 233-42; Athyros VG, Kakafika AI, Tziomalos K, Karagiannis A, Mikhailidis DP (July 2008). "Antisense technology for the prevention or treatment of cardiovascular disease: what's the next breakthrough new drug?" Expert Opin Investig Drugs 17 (7): 969-72. It is administered by injection once a week. The compound is a "next generation" antisense oligonucleotide; the nucleotide is bound by a phosphorothioate bond rather than a phosphodiester bond between RNA and DNA, and the sugar moiety is the deoxyribose in the middle portion of the molecule and its ends. 2'-O-methoxyethyl modified ribose. These modifications allow weekly dosing of the drug's resistance to degradation by the nuclease. The drug accumulates in the liver, which is convenient because apolipoprotein B acts predominantly there. The complete sequence is
G ^* -C ^* -C ^* -U ^* -C ^* -d A -dG-d T -dC-dT-dG-d mC-dT-dT- dmC -G ^* -C ^* -A ^* -C ^* - C ^* [d = 2'-deoxy, ^* = 2'-O- (2-methoxyethyl)] (having a 3'→ 5'phospholothioate bond). Phase III trials were conducted in patients with both familial hypercholesterolemia (FH), homozygotes (ho) and heterozygotes (he). FH is an exceptionally high-density lipoprotein cholesterol-causing hereditary disease. Both trials showed the highest efficacy ever seen in those two patient populations, and exceptional performance with a relatively low dropout rate compared to other injectable agents.

Duchenne muscular dystrophy: In Duchenne muscular dystrophy (DMD), the patient's muscle cells are destroyed and lost, leading to progressive weakness and death. AVI-4658 is a targeted antisense therapy that restores the expression of major proteins deficient in patients with dystrophin, Duchenne muscular dystrophy. Results from an open-label Phase II dose escalation study of 19 patients showed that muscle biopsies were taken from each patient when treatment was completed. The team discovered that the patient's ability to produce functional mRNA through "exon skipping" was restored with the use of AVI-4658, and ultimately allowed them to make functional dystrophin proteins. did.

Myotonic dystrophy: Myotonic dystrophy is the most common muscle disease in adults that primarily affects skeletal muscle, heart and nervous system. It is caused by a mutation that causes many repeats of the three letters of the genetic code (CTG) in a gene called DMPK. That is, messenger RNA produced from a mutant gene also contains anomalous repeat elongation that causes the RNA to accumulate in the cell nucleus. There, it sequesters and blocks the function of a protein called Muscleblind-like1 and activates another protein called CELF1. These proteins antagonize another protein, resulting in aberrant expression of the protein from many other genes in adult tissues, leading to disease. To counter this, Cooper and colleagues explore the aberrant RNA repeat moiety and antisense, a simple strand of genetic material that targets ribonuclease H for the harmful RNA responsible for its degradation. Produced an oligonucleotide. It has also been reported that the effect can be enhanced when the antisense oligonucleotide is used in combination with other antisense oligonucleotides that aid in the release of the isolated Muscleblind-like1.

Diabetes: SGLT2 (ISIS388626) is an antisense drug that achieves a decrease in sodium-glucose cotransporter 2 (SGLT2) levels that results in a significant decrease in blood glucose levels in diabetic patients.

Hepatitis: Persistent hepatitis C virus (HCV) infection is a major cause of liver disease. Antisense oligonucleotides are a promising classification of antiviral drugs. Such agents under development include ISIS14803, a 20-unit phosphorothioate oligonucleotide that inhibits HCV replication and protein expression.

Therapeutic Methods The oligonucleotides provided herein and their compositions are useful as therapeutic agents for a variety of medical conditions, including their use as antiviral agents. In some embodiments, the oligonucleotides provided can be used as therapeutic agents for diseases through the regulation of DNA and / or RNA activity. In some embodiments, the oligonucleotides provided can be used to inhibit specific gene expression. For example, the oligonucleotide provided may be complementary to a specific target messenger RNA (mRNA) sequence. It can be used to inhibit viral replication of various viruses. Examples of virus families include orthomixovirus, poxvirus, herpesvirus, papillomavirus, picornavirus, flavivirus, retrovirus, hepatitis virus, paramyxovirus, leovirus, parvovirus, phyllovirus, coronavirus, Includes arenaviruses, rabdoviruses and adenoviruses. Further known are viral families, which are contemplated herein. Other examples include use against HIV RNA or other retroviral RNA, or as an antisense compound for hybridizing to the HIV mRNA encoding the tat protein, or to the TAR region of the HIV mRNA. In some embodiments, the nucleic acid mimics the secondary structure of the TAR region of HIV mRNA, thereby binding to the tat protein. In some embodiments, the provided oligonucleotide does not, or only minimizes, inhibits the expression of other proteins in the cell by contacting the cell with the provided oligonucleotide of the target protein. Used to inhibit expression. In some embodiments, target protein inhibition occurs in vivo in mammals. In some embodiments, the therapeutic effect size of the oligonucleotide provided is administered to inhibit target protein expression.

Other examples of proteins whose expression can be regulated include Jun-N terminal kinase (JNK) protein, diacylglycerol acyltransferase I, apolypoprotein B, glucagon receptor, aurora B, acyl CoA cholesterol acyltransferase 2, C-reactive. Included are proteins, the STAT (Signal Kinase and Transcription Activator) family of proteins, and MDR P glycoproteins. In some embodiments, the oligonucleotides provided can be used to inhibit protein phosphatase 1B (PTP1B) expression, RNA-dependent RNA viral polymerase. In some embodiments, the oligonucleotides provided can be used to induce events such as apoptosis in cancer cells, or to make cells more susceptible to apoptosis. In some embodiments, the oligonucleotides provided can be used to regulate protein activity. In some embodiments, the oligonucleotides provided may aid in the regulation of ribonuclease H activity that targets multidrug resistant (MDR) RNA molecules.

In some embodiments, the invention provides a method of treating a disease mediated by unwanted gene expression in a subject in need of such treatment (eg, a mammal such as a human). "Disease" means a disease or symptom. The method comprises, for example, administering to the subject an effect size of the oligonucleotide provided.

Examples of diseases mediated by unwanted gene expression include cancer (eg, leukemia, tumor, and metastasis), allergies, asthma, obesity, inflammation (eg, inflammatory diseases such as inflammatory respiratory disease syndrome), Hypercholesterolemia, blood disorders, severe acute respiratory syndrome (SARS), obstructive airway disorders, asthma, autoimmune disorders, retroviral disorders such as AIDS or HIV, other viral infections, intrauterine infections, Metabolic diseases, infections (eg, bacterial, viral, yeast, fungal), central nervous system diseases, brain cancer tumors, degenerative nerve diseases, cardiovascular diseases, and angiogenesis-related diseases, angiogenesis, and angiogenesis. Will be.

In some embodiments, the oligonucleotides provided are useful for cancer therapeutic applications, including pancreatic cancer and other diseases or disorders with abnormal cell proliferation.

Located in the upper abdomen (inside the retroperitoneum), the pancreas is a dual-function gland of the digestive and endocrine systems. In some cases, the pancreas functions as an endocrine gland (eg, producing some important hormones). In some cases, the pancreas functions as an endocrine gland (eg, a secretion containing digestive enzymes that passes through the small intestine).

Pancreatic cancer is the fourth most common cause of cancer death in the United States (after lung, colon and breast cancer) and accounts for 6% of all cancer-related deaths. In 2008, an estimated 37,680 new cases of pancreatic cancer will be diagnosed in the United States and 34,290 will die. Incidence is only a decisive risk factor of smoking (smokers are four times more likely to develop the disease than nonsmokers) and increase linearly at age 50. Invasive pancreatic cancer is almost always fatal. The median survival time for all patients is 4-6 months. The relative 1-year survival rate is 24%; the overall 5-year survival rate is less than 5%.

Pancreatic cancer is early asymptomatic and often remains undiagnosed for several months (less than one-third of patients are diagnosed within 2 months of onset). In some cases, delayed diagnosis results in (partially or wholly) cancerous cells metastasizing to the liver or lymph nodes.

Currently, surgery (pancreatic resection) is the primary and only curative treatment for pancreatic cancer. However, at the time of diagnosis, only 15-25% of the tumor can be resected, and only 10-20% of patients undergoing surgery survive for more than 2 years. Once tumor wetting occurs and other tissues are affected, surgery is no longer possible.

In some cases, diabetes or pancreatitis can cause an individual to become ill and develop a proliferative disorder of multiple pancreatic cells. In some cases, an individual is at risk of developing multiple pancreatic cell proliferative disorders due to a hereditary syndrome selected from the group consisting of hereditary nonpolyposis colorectal cancer (HNPCC) and familial adenomatous polyposis (FAP). It is increasing. In some cases, individuals are at increased risk of developing multiple pancreatic cell proliferative disorders due to gene mutations selected from the group consisting of MSH2, MSH6, MLH1, and APC.

Ideally, effective treatment of pancreatic cancer should (i) suppress early tumor mass initially and subsequently and (ii) treat metastatic tumor cells. Chemoprevention (drug administration of drugs, biologics, nutritional agents, etc.) indicates, nullifies, or inhibits the progression of carcinogenesis, thereby reducing the risk of developing a wet or clinically significant disease.

In certain embodiments, the present specification discloses methods of treating pancreatic cancer. As used herein, "pancreatic cancer" includes the form of cancer of the pancreas. In some embodiments, the pancreatic cancer is metastatic pancreatic cancer. In some embodiments, pancreatic cancer is a carcinoma, sarcoma, cancer, or a combination thereof. In some embodiments, the pancreatic cancer being treated includes sporadic and hereditary pancreatic cancer. In some embodiments, the pancreatic cancer is ductal cell carcinoma, acinic cell carcinoma, papillary mucinic cancer, ring cancer, glandular squamous cell carcinoma, undifferentiated cancer, mucinic cancer, giant cell carcinoma, small cell carcinoma, cyst. Cancer, serous cyst cancer, mucinous cyst cancer, unclassified pancreatic cancer, pancreatic blastoma, or a combination thereof.

In some embodiments, an individual in need of treatment for pancreatic cancer has symptoms of a local tumor of the pancreas. In some embodiments, individuals in need of treatment for pancreatic cancer have symptoms of negative regional lymph node biopsy. In some embodiments, individuals in need of treatment for pancreatic cancer have symptoms of nodular negative pancreatic tumors (eg, negative lymph node metastases). In some embodiments, individuals in need of treatment for pancreatic cancer have symptoms of nodular positive tumors (eg, positive lymph node metastases).

In some embodiments, pancreatic cancer in an individual in need of treatment for pancreatic cancer has spread elsewhere in the body. In some embodiments, pancreatic cancer has spread to a location selected from the group consisting of lymph nodes, stomach, bile ducts, liver, bones, ovaries, peritoneum and brain.

In some embodiments, cancer cells or precancerous cells are identified by tissue classification or categorization of tissue samples (eg, biopsy samples). In some embodiments, cancer cells or precancerous cells are identified through the use of appropriate molecular markers.

In some embodiments, pancreatic cancer in individuals requiring treatment for pancreatic cancer is graded according to the American Joint Commission on Cancer (AJCC) TNM classification system, where the tumor (T) is staged Tx, T1, T2, Classified as T3, T4; regional lymph nodes (N) are classified as stage of NX, N0, N1; distant metastases (M) are classified as stage of MX, M0, or M1. In some embodiments, pancreatic cancer in individuals requiring treatment for pancreatic cancer is staged 0, I, IA, IB, II, IIA, IIB, III, and IV pancreatic cancer. In some embodiments, pancreatic cancer in an individual requiring treatment for pancreatic cancer is graded into malignancy GX (eg, malignancy cannot be assessed), malignancy 1, malignancy 2, malignancy 3 or malignancy 4. Will be done.

More specific examples of cancers treated with the provided oligonucleotides include breast cancer, lung cancer, melanoma, colorectal cancer, bladder cancer, ovarian cancer, prostate cancer, renal cancer, squamous cell carcinoma, glioma, Examples include Kaposi's sarcoma, multiple myeloma, and leukemia.

Evaluation and Treatment of Cancer The term "tumor cell antigen" is defined herein as an antigen that is present in higher amounts in tumor cells or fluid than in unrelated tumor cells, normal cells, or normal fluid. The presence of the antigen can be tested by a number of assays well known to those of skill in the art, including positive and / or negative selection with antibodies such as ELISA assay, radioimmunoassay, or Western blotting. Not limited to this.

An "apoptosis-inducing agent" is defined herein as inducing apoptosis / programmed cell death, eg, an anticancer drug and a treatment in which cells (eg, tumor cells) are induced to undergo programmed cell death. including. An exemplary apoptosis inducer is described in more detail below.

The term "apocalyptic" or "programmed cell death" refers to a biological process in which unwanted or useless cells are removed during development and other normal biological processes. Apoptosis is a form of cell death that occurs under normal biological conditions, in which the cell actively participates in its own demise (“cell suicide”). It is most often found during normal cell rotation and tissue homeostasis, embryogenesis, induction and maintenance of immune tolerance, nervous system development and endocrine-dependent tissue atrophy. Apoptotic cells exhibit unique morphological and biochemical characteristics. These features include chromatin condensation, nuclear and cytoplasmic condensation, cytoplasmic and nuclear division into membranes bound to vesicles containing ribosomes, and morphologically intact mitochondrial and nuclear material. In vivo, these apoptotic bodies are immediately recognized and phagocytosed by macrophages, dendritic cells or adjacent epithelial cells. Thanks to this efficient mechanism of removing apoptotic cells in the body, an inflammatory response is not elicited. In vitro, not only the remaining cell fragments, but also the apoptotic bodies are extremely swollen and finally lysed. This terminal process of in vitro cells is named "secondary necrosis". Apoptosis can be measured by methods well known to those of skill in the art such as DNA fragmentation, exposure to Annexin V, activation of caspases, release of cytochrome c, and the like. Death-induced cells are referred to herein as "apoptotic cells." Apoptosis can also be tested using a standard annexin V apoptosis assay: NIH: OVCAR-3 cells grown on a 6-well plate (NUNC) and irradiated or antagonistic (or in combination with another anticancer drug) 4 Treat for ~ 48 hours, wash and stain with Annexin V-FITC (BD Fermingen) for 1 hour. Cells are analyzed by flow cytometry (CellQuest, Becton Dickinson), counterstained with propidium iodide, and analyzed again by flow cytometry.

Patients may be evaluated for symptoms at one or more time points, including before, during, and after the treatment regimen. Treatment results in amelioration of the subject's symptoms and one or more of the following factors: tumor shrinkage, cell proliferation reduction, cell proliferation reduction, angiogenesis reduction, apoptosis increase, or at least some of the tumor cells. It can be assessed by determining if a decrease in survival has occurred. In some cases, when one or more of these occur, the result may be partial or total elimination of the cancer and prolongation of the patient's survival. Alternatively, in end-stage cancer, treatment can result in a lull of illness, improved quality of life and / or prolonged survival.

Cell Migration Assay Methods Cell migration assays are described in the literature, eg, by Brooks, et al., J. Clin. Invest 1997, 99: 1390-1398, and methods for measuring cell migration are well known to those of skill in the art. be. In one method of measuring cell migration described herein, the membrane from the transwell migration chamber is coated with a substrate, the transwell is washed, and the non-specific binding site is blocked with BSA. Tumor cells are harvested from subconfluent cultures, washed and resuspended in migration buffer with or without assay antibody. After allowing the tumor cells to migrate to the back surface of the coated transwell membrane, the cells remaining on the upper surface of the membrane are removed, and the cells migrating to the back surface are stained with crystal violet. Then, cell migration is directly quantified by the number of cells per microscopic field of view.

Tumor Growth Assay Methods Tumor growth can be assayed by methods well known to those of skill in the art, such as SCID mouse models, nude mouse models, and BALB / c mice with syngeneic tumors. A SCID mouse model for tumor growth is performed as follows: Subconfluent human M21 melanoma cells (or any desired tumor cell type) are harvested, washed and placed in sterile PBS (20x106 / mL). Resuspend. SCID mice are subcutaneously injected with 100 μL of M21 human melanoma cell (2x106) suspension. On the third day after tumor cell injection, mice are treated intraperitoneally with an untreated or intraperitoneal antagonist of the desired dose range. The mice are treated daily for 24 days. Tumor size is measured with a caliper and the volume is estimated using the formula V = (LxW2) / 2 (where V is the volume, L is the length, and W is the width).

Alternatively, nude mouse models, SCID mouse models and / or BALB / c syngeneic mouse models can also be used to assess tumor growth and its inhibition by the humanized anti-endoglin antibodies or antigen-binding fragments described herein. can do.

Cell proliferation assay method Cell proliferation can be assayed by methods well known to those of skill in the art. As described herein, subconfluent human endothelial cells (HUVEC) are resuspended from ECV or ECVL cells in a growth buffer containing low (5.0%) serum with and without CM (25 μL). It becomes cloudy and can proliferate endothelial cells for 24 hours. Proliferation is quantified by measuring mitochondrial dehydrogenase activity using a commercially available WST-1 assay kit (Chemicon). Also, as described herein, growth can be quantified by measurement of 3H uptake using standard methods. (She et al., Int. J. Cancer, 108: 251-257 (2004)).

Other methods of cell proliferation assessment are well known in the art and are contemplated herein. In addition, unrestricted examples are described in more detail in the Examples.

The classification and staging systems described herein are meant to evaluate the cancer treatments described herein; in addition, other staging schemes are well known in the art and are described in this article. It should be understood that it can be used in conjunction with the methods described herein. As an example, the TNM classification of malignant tumors can be used as a cancer staging system to account for the extent of cancer in a patient. T describes the tumor size and whether it invades adjacent tissue, N describes the regional lymph nodes involved, and M describes the distant metastasis. TNM is maintained by the Union for International Cancer Control (UICC) and has been adopted by the American Joint Commission on Cancer (AJCC) and the International Federation of Gynecologic Obstetrics and Sciences (FIGO). Not all tumors, for example brain tumors, have a TNM classification. Generally, T (a, is, (0), 1-4) is measured as the size or direct extent of the primary tumor. N (0-3) represents the degree of diffusion to the regional lymph node: N0 means that the tumor cells are not in the regional lymph node; N1 is the closest or minority tumor cell to the regional lymph node. Means that the tumor cells have spread to between N1 and N3; N3 means that the tumor cells have spread to the most distant or multiple regional lymph nodes. It means that you are doing it. M (0/1) represents the presence of metastases: M0 means no distant metastases; M1 means that metastases are occurring in distant organs (beyond regional lymph nodes). .. Other parameters can also be evaluated. G (1-4) represents the malignancy of cancer cells (ie, if they look similar to normal cells, they are low grade, and if they look poorly differentiated, they are high grade. Is). R (0/1/2) represents the integrity of the surgery (ie, the presence or absence of cancer cells at the resection border). L (0/1) represents infiltration into the lymphatic vessels. V (0/1) represents infiltration into the vein. C (1 to 4) represents a certainty (quality) factor of V.

Degrading, inhibiting the growth of a cancer cell, comprising contacting the cell with an amount of a compound described herein that is effective for degrading, inhibiting or killing the cancer cell herein. Or a method for dying is provided.

In the present specification, the effect amount of the compound described in the present specification is administered to an individual in order to suppress the increase in tumor size, reduce the size of the tumor, reduce the growth of the tumor or prevent the growth of the tumor. Methods are provided for suppressing tumor size growth, reducing tumor size, reducing tumor growth or preventing tumor growth. In some cases, tumor treatment involves a relief of symptoms, i.e., treatment of the patient does not exacerbate the cancer and prolongs the survival of the patient.

Patients may be evaluated for symptoms at one or more time points, including before, during, and after the treatment regimen. Treatment results in amelioration of the subject's symptoms and determines whether one or more of the following events: tumor shrinkage, decreased tumor cell proliferation, decreased cell number, decreased angiogenesis and / or increased apoptosis. Can be evaluated by doing. In some cases, when one or more of these occur, the result may be partial or total elimination of the cancer and prolongation of the patient's survival. Alternatively, in end-stage cancer, treatment can result in a lull of illness, improved quality of life and / or prolonged survival.

Other methods of assessing treatment are well known in the art and are contemplated herein. In an exemplary embodiment, the pro-oligonucleotides of the invention are such as mammals (eg, humans) suffering from a medical disorder, eg, cancer, or a non-malignant condition characterized by the presence of an unwanted cell classification. Administered to the subject.

The primary outcome scale can be assessed in patients treated using the methods described herein, including, for example, progression-free survival. In one embodiment, the increase in progression-free survival is observed in an amount of about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or more as compared to the case without treatment. In another embodiment, the increase in progression-free survival is about 3 months, about 6 months, about 9 months, about 12 months, about 18 months, about 2 years, about 3 years, as compared to the case without treatment. Survival increases for about 4 years, about 5 years or more.

Secondary outcome measures can also be evaluated, including duration of response, tumor progression time, overall survival, severe and non-serious adverse events. For example, treatment can prevent (ie, lull) or improve the progression of the disease. Alternatively, or in addition, other objectives may be measured with respect to one or more of the following: reduced tumor mass, reduced angiogenesis, reduced side effects, reduced adverse reactions, and / or improved patient compliance.

Other specific examples of diseases or disorders for which treatment with the compounds or compositions of the invention are useful for treatment or prevention include transplant rejection (eg, kidneys, liver, heart, lungs, island cells, pancreas, bones, etc. Bone marrow, corneum, small intestine, skin allogeneic or heterologous transplant and other implants), implant vs. host disease, osteoarthritis, rheumatoid arthritis, multiple sclerosis, diabetes, diabetic retinopathy, inflammatory bowel disease ( For example, Crohn's disease, ulcerative colitis, and other intestinal diseases), renal disease, malaise, septic shock, lupus, severe myasthenia, psoriasis, dermatitis, eczema, seborrhea, Alzheimer's disease, Parkinson's disease. , Chemotherapeutic stem cell protection, in vitro selection or purification for autologous or allogeneic bone marrow transplantation, intraocular disease, retinopathy (eg, yellow spot degeneration, diabetic retinopathy, and other retinopathy), corneal disease , Glaucoma, infectious diseases (eg, bacterial, viral, or fungal), including, but not limited to, heart disease including, but not limited to, restenosis.

Activation of Ribonuclease L The 2'-5'oligoadenylation (2-5A) / ribonuclease L pathway is one of the interferon-induced enzymatic pathways. Ribonuclease L is activated after binding to a 5'-phosphorylated fragment of 2'-5'adenylic acid. These fragments of 2'-5'adenylic acid (2-5A) are produced under the control of 2'-5' oligo (A) synthetase. This pathway is part of the innate immune system and has an important role in the prevention of viral infections. 2-5A-induced cleavage of single-stranded RNA results in apoptosis. In vivo stable phosphorothioate analogs of 2-5A have been shown to be potent activators of ribonuclease L (Xianh et al., Cancer Research (2003), 63: 6795-6801). In this study, the 2-5A analog induced ribonuclease L activity and caused in-culture apoptosis of end-stage metastatic human prostate cancer cell lines DU145, PC3 and LNCaP.

Persistent activation of ribonuclease L initiates an apoptotic mitochondrial pathway that eliminates virus-infected cells as well as cancerous / tumor cells. Ribonuclease L can inhibit fibrosarcoma growth, prostate cancer growth, colorectal cancer growth and pancreatic cancer growth. Given the common role of ribonuclease L in various cancers, it is contemplated that the invention described herein can be used in the treatment of any type of cancer. Silverman, RH, Cytokine Growth Factor Rev, 18 (5-6): 381-388 (2007); Bisbal, C. and Silverman, RH, Biochimie. 89 (6-7): 789-798 (2007). As an example, downregulation of ribonuclease L reduces either the expression level of either the gene (s) or gene (s) encoding ribonuclease L, as compared to a predetermined level of ribonuclease L in a typical healthy population, ribonuclease L. Silencing the gene (s) or genes (s) that encode the gene, reducing the level of expression / translation of proteins containing ribonuclease L, reducing the amount of ribonuclease L present in the cell, and / or the activity of ribonuclease L. Represents any decline. Alternatively, a decrease in any of the ribonuclease L levels described herein may indicate downregulation of ribonuclease L.

In some embodiments, the oligonucleotides provided are useful for the treatment of diseases with downregulated ribonuclease L. In some embodiments, the disease associated with the downregulated ribonuclease L is cancer. In some embodiments, the cancer is pancreatic cancer, prostate cancer, or colorectal cancer. Alternatively, the oligonucleotides provided herein are useful for the treatment of diseases with upregulated ribonuclease L. In some embodiments, the disease with the upregulated ribonuclease L is chronic fatigue syndrome. Further diseases with up-expressed ribonuclease L are well known in the art and are discussed herein.

When used therapeutically, the oligonucleotides provided are administered as a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a provided oligonucleotide containing a pharmaceutically acceptable salt thereof, or a therapeutic effect size of the pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof. It contains a diluent, a pharmaceutically acceptable excipient, and at least one pharmaceutically acceptable inert ingredient selected from a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition is formulated for intravenous injection, oral administration, oral administration, inhalation, intranasal administration, topical administration, ocular administration or ear administration. In a further embodiment, the pharmaceutical composition comprises tablets, pills, capsules, solutions, inhalants, nasal drops, suppositories, suspensions, gels, colloids, dispersants, suspensions, solutions, Emulsions, ointments, lotions, eye drops or ear drops.

Pharmaceutical Compositions When used therapeutically, the oligonucleotides or oligonucleotide compositions provided herein are administered as pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises a therapeutic effect size of the provided oligonucleotide, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, pharmaceutically acceptable excipient. It contains a form and at least one pharmaceutically acceptable inert ingredient selected from pharmaceutically acceptable carriers. In some embodiments, the pharmaceutical composition is formulated for intravenous injection, oral administration, oral administration, inhalation, intranasal administration, topical administration, ocular administration or ear administration. In some embodiments, the pharmaceutical composition comprises tablets, rounds, capsules, solutions, inhalants, nasal drops, suppositories, suspensions, gels, colloids, dispersants, suspensions, Liquids, emulsions, ointments, lotions, eye drops or suppositories.

In some embodiments, the invention provides a pharmaceutical composition comprising a chiral controlled oligonucleotide, or a composition thereof mixed with a pharmaceutically acceptable excipient. Those skilled in the art will recognize that the pharmaceutical composition comprises a pharmaceutically acceptable salt of the chiral controlled oligonucleotide described above, or a composition thereof.

Compounds that Enhance and Target Delivery The oligonucleotides provided herein can be delivered using various delivery strategies, including nucleic acid complexes with various ligands, as well as the use of nanocarrier approaches. Both nucleic acid delivery strategies contemplate the use of the oligonucleotides provided herein. The choice between typical delivery strategies, including but not limited to chemical complexes, cationic lipid / liposome transport media and supramolecular nanocarriers, depends on the context of the treatment and how to determine the optimal delivery mode. , Well known to those of skill in the art, and will be discussed further herein.

Cell-penetrating compound (“CPC”)
It is known that a large number of compounds act as carriers for loads such as nucleic acids and promote the influx of nucleic acids into cells set in vivo. An exemplary carrier is described in Dietz et al., Molecular & Cellular Neuroscience, 27 (2): 85-131 (2004), which is incorporated herein by reference. The Tat and Antennapedia transcriptional regulator-derived prototype CPCs are linked by the majority of new parts. As an example, the peptide CPC can be a relatively short (9-30 amino acid) polycationic peptide high in arginine and lysine, or a membrane-interacting hydrophobic sequence. CPCs can be bound or chemically bound to peptides, oligonucleotides or nanocarriers by recombinant DNA technology, and then include a "load" of CPCs.

Cell Target Ligand (“CTL”)
Another strategy is to deliver oligonucleotides by using CTLs that bind with high affinity to cell surface receptors capable of efficient internalization. Promising ligands include antibodies, polypeptides from the phage display library, and small organic molecules. Further cell-targeted ligands will be known or developed by those of skill in the art and are intended for use in the present invention as described herein (for ASGPR-GalNAc complex siRNAs and oligonucleotides, eg. , International Publication WO 201207254 A1). Since various receptors are often selectively expressed in a particular cell type, this approach offers improved selectivity for the oligonucleotide reagent. Examples of receptor targets include, but are not limited to, lipoprotein receptors (such as those in the liver), integrins, receptor tyrosine kinases, and G protein-coupled receptor (GPCR) superfamily.

Nanocarriers Various supramolecular nanocarriers can be used for the delivery of nucleic acids. Examples of nanocarriers include, but are not limited to, liposomes, cationic polymer complexes and various macromolecules. Complex formation of various polycations and nucleic acids is another approach for cell-to-cell delivery; it is a PEGylated polycation, a polyethyleneamine (PEI) complex, a cationic block copolymer, and a dendrimer. Use is mentioned. Several cationic nanocarriers, including PEI and polyamide amine dendrimers, aid in the release of contents from endosomes. Other approaches include the use of polymer nanoparticles, polymer micelles, quantum dots and lipoplexes.

Further nucleic acid delivery strategies are known in addition to the exemplary delivery strategies described herein.

For therapeutic and / or diagnostic applications, the compounds of the invention may be formulated for a variety of administration method applications, including systemic and topical or localized administration. Techniques and formulations are commonly found in Remington, The Science and Practice of Pharmacy, (20th ed. 2000).

The oligonucleotides provided, and their compositions, are effective over a wide range of doses. For example, in adult treatment, examples of doses where about 0.01 to about 1000 mg, about 0.5 to about 100 mg, about 1 to about 50 mg per day, and about 5 to about 100 mg per day can be used. Is. The exact dose will depend on the route of administration, the form in which the compound is administered, the subject to be treated, the weight of the subject to be treated, and the preferences and experience of the attending physician.

Pharmaceutically acceptable salts are generally well known to those of skill in the art and include, for example, acetates, benzenesulfonates, besilates, benzoates, bicarbonates, tartrates, bromides, calcium edetate, etc. Carnsylate, carbonate, citrate, edetate, edicylate, estrate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolylarsanylate, hexylresolcinate , Hydrabamine, hydrogen bromide, hydrogen chloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, mucilate, napcil Acids, nitrates, pamoates (pamoates), pantothenates, phosphates / diphosphates, polygalacturonates, salicylates, stearate, basic acetates, succinates, sulfates, Examples include, but are not limited to, tannate, tartrate, or theocrousate. Other pharmaceutically acceptable salts can be found, for example, in Remington, The Science and Practice of Pharmacy (20th ed. 2000). Preferred pharmaceutically acceptable salts include, for example, acetate, benzoate, bromide, carbonate, citrate, gluconate, hydrogen bromide, hydrogen chloride, maleate, mesylate, napcilic acid. Examples include salts, pamoates (embonates), phosphates, salicylates, succinates, sulfates, or tartrates.

Depending on the particular sign to be treated, such agents may be prescribed in liquid or solid dosage form and administered systemically or topically. The agent may be delivered, for example, in a timed or sustained low release form well known to those of skill in the art. Formulation techniques and administration can be found in Remington, The Science and Practice of Pharmacy (20th ed. 2000). Suitable routes are oral, oral, inhalation spray, sublingual, intrarectal, transdermal, transvaginal, transmucosal, nasal or intestinal administration; intramuscular, subcutaneous, subarachnoid space, direct brain (heart) Intravenous, intravenous, joint, intrathoracic, intrathecal, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular as well as parenteral delivery, including intrathecal injection, or others Delivery method may be mentioned.

For injection, the agents of the invention can be formulated and diluted in aqueous solutions such as in Hanks' solutions, Ringer's solutions, or physiologically compatible buffers such as saline. For such transmucosal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally well known in the art.

The use of a pharmaceutically acceptable inert carrier for prescribing the compounds in the present specification disclosed for the practice of the invention in a suitable formulation for systemic administration is within the scope of the invention. With proper selection of carriers and proper manufacturing practices, the compositions of the invention, in particular these formulations of solutions, can be administered parenterally, such as by intravenous injection.

The compound can be readily formulated in a formulation suitable for oral administration using a pharmaceutically acceptable carrier well known to those of skill in the art. Such a carrier prepares the compound of the present invention as a tablet, a pill, a capsule, a liquid, a gel, a syrup, a slurry, a suspension, etc. for oral ingestion of a subject to be treated (for example, a patient). enable.

For nasal or inhalation delivery applications, the agents of the invention may also be formulated by methods well known to those of skill in the art, eg, solubilizing, diluting or dispersing substances such as saline, preservatives such as benzyl alcohol, absorption. Examples include, but are not limited to, accelerators and fluorocarbons.

Suitable pharmaceutical compositions for use in the present invention include compositions in which the active ingredient is contained in an effect size to achieve its intended purpose. The determination of effect size can be adequately made by one of ordinary skill in the art, especially in light of the detailed disclosure provided herein.

In addition to the active ingredient, these pharmaceutical compositions include suitable pharmaceutically acceptable carriers containing excipients and auxiliaries that facilitate the process of the active compound in the pharmaceutically usable formulations. Can include. The formulation prescribed for oral administration can be in the form of tablets, dragees, capsules, or liquids.

For the pharmaceutical product for oral use, the active compound is mixed with a solid excipient to obtain a tablet or a dragee core, if necessary, after adding an appropriate auxiliary agent, and the obtained mixture is ground. It may be obtained, and it may be obtained by a mixed processing of granules. Suitable excipients are sugars including lactose, sucrose, mannitol, or sorbitol, in particular; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanto gum, methylcellulose, hydroxypropylmethylcellulose, Fillers such as sodium carboxymethyl cellulose (CMC) and / or polyvinylpyrrolidone (PVP: povidone). If desired, a disintegrant may be added, such as crosslinked polyvinylpyrrolidone, agar, or a salt thereof such as alginic acid or sodium alginate.

The sugar coating core is provided with a suitable coating. A high concentration sugar solution may be used for this purpose, which may be arabic rubber, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG), and / or titanium dioxide, lacquer solution, and suitable organic solvent or It may contain a solvent mixture. Dyes or pigments may be added to the tablet or dragee coating to identify or characterize the active compound doses of the various combinations.

Pharmaceutical formulations that can be used orally include sealed soft capsules made of gelatin and plasticizers such as glycerol or sorbitol, as well as push-fit capsules made of gelatin. The push-fit capsule may be mixed in a mixture with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and a stabilizer, the active ingredient. May include. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin or liquid polyethylene glycol (PEG). In addition, stabilizers may be added.

Depending on the particular symptom or condition being treated or prevented, additional therapeutic agents that are normally administered to treat or prevent the symptom may be administered with the inhibitors of the invention. For example, chemotherapeutic agents or other antiproliferative agents can be used in combination with the inhibitors of the invention to treat proliferative disorders and cancers. Examples of well-known chemotherapeutic agents include, but are not limited to, adriamycin, dexamethasone, vincristine, cyclophosphamide, fluorouracil, topotecan, taxol, interferons, and platinum derivatives.

Other examples of agents, the non-lasemiprooligonucleotides of the invention, are, but are not limited to, anti-inflammatory drugs such as corticosteroids, TNF blocking drugs, IL-1 RA, azathiopurine, cyclophosphamide, and sulfasalazine. Immunomodulatory and immunosuppressive drugs such as cyclosporin, tachlorimus, rapamycin, mofetyl mycophenolate, interferons, corticosteroids, cyclophosphamide, azathiopurine, and sulfasalazine; acetylcholinesterase inhibitors, MAO inhibitors Neurotrophic factors such as, interferons, antispasmodics, ion channel blockers, lysole, and anti-Parkinson drugs; β blockers, ACE inhibitors, diuretics, nitrates, calcium channel blockers, And cardiovascular drugs such as statins; liver disease drugs such as corticosteroids, cholestiramines, interferons, and antiviral drugs; corticosteroids, anti-leukemia drugs, and growth factors It can also be used in combination with drugs for treating blood diseases such as insulin, drugs for treating diabetes such as insulin analogs, α-glucosidase inhibitors, biguanides, and drugs for improving insulin sensitivity; and drugs for treating immunodeficiency such as gamma globulin.

These additional agents may be administered separately from the chiral controlled oligonucleotides provided, and their compositions, as part of multiple dosing regimens. Alternatively, these additional agents may be a chiral-controlled oligonucleotide provided in a single composition, and a portion of a single dosage form mixed with the composition.

The examples and formulations provided below further illustrate and illustrate the oligonucleotides provided, their compositions, and methods of their manufacture. It is to be understood that the scope of the invention is by no means limited by the scope of the following examples and formulations.

The functions and advantages of these and other embodiments of the invention will be understood in more detail from the examples below. The following examples are intended to illustrate the benefits of the invention and do not illustrate the full scope of the invention.

Methods for Analyzing Oligonucleotide Compositions In some embodiments, the present invention provides methods for analyzing oligonucleotide compositions. In some embodiments, the provided method detects, determines, and / or quantifies, for example, the identification, purity, quantification and / or quality of one or more oligonucleotides. In some embodiments, the invention is
a) A step of performing a first analysis of a first composition comprising multiple different types of oligonucleotides; and b) the first analysis performed is a chiral-controlled composition of the oligonucleotide. It is a step of comparing the second analysis of the second composition under the same conditions as the first analysis, and the difference between the first and second analyzes is compared with the second composition. The present invention provides a method comprising a step of reflecting the difference in the presence or level of the first at least one oligonucleotide.

In some embodiments, the second composition comprises only a single type of the oligonucleotide. In some embodiments, the second composition comprises one or more types of the oligonucleotide. In some embodiments, the provided method is used, for example, for quality control of the oligonucleotide compositions shown in Examples 17 and 48. As will be appreciated by those skilled in the art, the data shown in Example 48 will be compared to the compositions shown in which the random oligonucleotide composition (the first composition) is a non-chiral controlled oligonucleotide synthesis. When synthesized by, it confirms that the chiral control composition (the second composition) is shown to contain specific oligonucleotide types such as total Rp or Sp at extremely low levels.

The aforementioned is a description of certain non-limiting embodiments of the invention. Therefore, it should be understood that the embodiments of the invention described herein merely illustrate the application of the principles of the invention. References herein to the details of the embodiments described are not intended to limit the scope of the claims.

Overview of oligonucleotide synthesis

As mentioned above, and as described herein, in some embodiments, the invention provides oligonucleotides and methods of their preparation. In some embodiments, the synthesis of oligonucleotides is carried out using a solid carrier. In some embodiments, the synthesis of oligonucleotides is done in solution. In some embodiments, the synthesis of oligonucleotides comprises the use of a solid carrier and the steps in a solution phase. In some embodiments, the step of using a solid support is performed on an oligonucleotide synthesizer.

Preparation of solid carrier:
A carrier (highly crosslinked polystyrene (HCP) or controlled porous glass (CPG)) was used to fill the first 5'-O-DMTr protected nucleosides contained in the oligonucleotide sequence. In some embodiments, the first nucleoside using an oxalyl group is described in Oxalyl-CPG: a labile support for synthesis of sensitive oligonucleotide derivatives, Nucleic Acids Research 1991, 19 (7), 1527 by Alul et al. 3'-OH group was attached to the amino group of the solid carrier. In some embodiments, a standard succinyl group was used as the linker.

Nucleotide detrityl on solid support:
A 3% TCA (trichloroacetic acid) dichloromethane (DCM, CH ₂ Cl ₂ ) solution was developed on a carrier-containing column placed in a synthesizer to deblock 5'-O-DMTr.

これらの研究で使用したキラルホスホラミダイトは以下の通りである：

さらなるホスホラミダイトは以下の通りである：

CMPT-mediated chiral phosphoramidite coupling:
The solid support is washed with anhydrous acetonitrile (ACN) and dried by reflux of dry argon, after which the free 5'-OH is bound to the next nucleotide in the oligonucleotide sequence and extended from the 3'end to the 5'end. I let you. As described in Wada et al., J. Am. Chem. Soc., 2008, 130, 16031-1603; Angew. Chem. Intern. Ed. 2009, 48, 496-499, this was accompanied by CMPT (below). It results in the co-expansion of the chiral phosphoramidite solution to a solid carrier, resulting in highly efficient coupling and excellent enantiomeric excess (typically> 99%), resulting in a chiral subphosphate diester product. Obtained. The solvent was usually acetonitrile (ACN), but other solvents could be used.

The chiral phosphoramidite used in these studies is:

Further phosphoramideites are:

Capping:
In some embodiments, the capping step was performed in two steps. In some embodiments, the above two capping steps were combined into one and performed in one pot. Unless otherwise stated, two-step capping was used in the synthesis of the examples described herein.

In 2-step capping, the solid support is washed with anhydrous ACN, dried by backflow of dry argon, and then usually containing a 5% Pac ₂ O solution in 2,6-lutidine / THF-1: 4 (v / v). The "Cap A" reagent was developed on a solid carrier. Although not intended to be limited by theory, Cap A was sufficiently reactive to efficiently cap (aculate) the free amine of the chiral auxiliary. In some embodiments, such protection prevents rearrangement / degradation of the intermediate phosphite ester. Immediately after this treatment, a 1: 1 mixture of "Cap A" and "Cap B" reagents was run onto a column containing a solid carrier. "Cap B" is a 16% N-methylimidazole (NMI) THF solution and when mixed with "Cap A" results in capping of unreacted 5'-OH oligonucleotides bound to, for example, a solid carrier. Although not intended to be limited by theory, without capping of the amino group of the chiral auxiliary group, for example, due to an unwanted intermolecular nucleophilic attack on the phosphorus atom of the free amine of the auxiliary group, The capping step was very important in that the sulfide step within the cycle caused the cleavage of internucleotide bonds and the degradation of oligonucleotides. In addition, uncoupling spilled with respect to obtaining high-purity crude full-length oligonucleotides, avoiding low yields and difficulties due to accumulation and redundancy of defective sequence elongation, if final purification is not impossible. Capping of the 5'-OH group of the solid-supported oligonucleotide shortmer was very important.

Sulfurization:
The solid support was washed with anhydrous ACN, dried by backflow of dry argon, and then the resulting capped phosphite triester intermediate was subjected to oxidative sulfurization. The sulfurizing agent solution was prepared by mixing, for example, a sulfurizing agent which is an alkyl thiosulfonic acid derivative (300 mM) and N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) (100 mM) in ACN. This was then developed on a column containing a solid carrier and reacted for a specific period of time (usually 5-60 minutes). In some embodiments, the thiosulfonate reagent of the sulfurizing agent was used as a 300 mM solution in anhydrous ACN without the addition of BSTFA. In some embodiments, reagent solutions with BSTFA and reagent solutions without BSTFA produced similar results.

Oxidation:
In some embodiments, instead of the sulfurization step, oxidation to the phosphodiester was performed. A 0.02 MI ₂ solution in a THF / pyridine / water (70: 20: 10-v / v / v) co-solvent system was developed to achieve oxidation and form non-chiral phosphodiester bonds.

5'-deblocking of DMTr groups:
Removal of DMTr was performed using 3% TCA in DCM. After deblocking, then coupling, capping, sulfurization / oxidation (either sulfurization with the same sulfid, or sulfurization with different sulfids, or oxidation instead of sulfurization for each of the next cycles) and The cycle can be advanced to further repeated rounds of deblocking. Alternatively, upon reaching the pre-designed length, the oligonucleotide was subjected to cycle exit and optionally post-synthesis treatment. In some embodiments, the oligonucleotide was removed prior to deblocking the terminal 5'-O-DMTr group.

Spontaneous removal of chiral auxiliary:
Spontaneous removal of chiral auxiliary was achieved during sulfidation, oxidation, deblocking of DMTr groups, or a combination thereof. No specific steps were required to remove the chiral auxiliary.

Cleavage and deprotection:
The oligonucleotide of the desired length was removed from its solid support by cleaving the corresponding linker (oxalyl or succinyl-bound HCP or CPG) and at the same time deprotecting the protecting group of the oligonucleotide. In some embodiments, solid-supported oligonucleotides are first dehydrated ACN-trimethylsilyl chloride-16, as described in Reese and Yan's J. Chem. Soc., Perkin Trans. 1, 2002, 2619. Anhydrous 1M 1,5-diazabicyclo (4.3.0) nona-5-en (DBN) or 1,8-diazabicycloundeca-7-en (DBU) solution in 1 (v / v). It was treated at room temperature for 10 minutes using and then washed with dehydrated CN. In some embodiments, the material was treated with a dehydrated pyridine solution of dehydrated propylamine (usually a 1: 4 ratio) at room temperature for 18 hours or at 60 ° C. for 2 hours. In some embodiments, both conditions yielded homogeneous crude compounds. In some embodiments, crude oligonucleotides were cleaved from the carrier and deprotected by treatment with 28% aqueous ammonia for 18 hours at room temperature or 5 hours at 60 ° C. In some embodiments, both conditions yielded homogeneous crude compounds. The solvent is then evaporated and the residue is treated with an aqueous solution of about pH 1.5 (pH may be varied if desired) mixed with 0-50% DMSO to give the crude product HPLC and UPLC / MS. It was attached to the analysis by the combination of. The product was purified by one or a combination of reverse phase HPLC (RP-HPLC), normal phase HPLC, ion exchange HPLC (IE-HPLC) or molecular sieving chromatography. In some embodiments, oligonucleotides were removed from the carrier, deprotected and purified prior to deblocking the DMTr group.

Example 1: Synthesis of Sulfide and Synthesis of Phosphoramideite

（ｉ）ＭｓＣｌ、ＮＥｔ_３；（ｉｉ）ＮａＭＴＳ；（ｉｉｉ）ＰｉｖＣｌ、ＮＥｔ_３；（ｉｖ）ＮａＭＴＳ、ＮａＩ；（ｖ）化合物４；（ｖｉ）ＴＭＳＣｌ、ＮＥｔ_３；（ｖｉｉ）化合物３５、ＤＥＡＤ、ＰＰｈ_３；（ｖｉｉｉ）ＴＢＡＦ；（ｉｘ）ＴｓＣｌ、ピリジン；（ｘ）Ａｃ_２Ｏ、ピリジン；（ｘｉ）ＫＴＴＳ；（ｘｉｉ）（ＣＯＣｌ）_２；（ｘｉｉｉ）Ｆｍｏｃ‐ＯＳｕ、Ｐｙ；（ｘｉｖ）ＮａＯＨ（ａｑ）；（ｘｖ）Ｎａ‐ｐ‐ＣｌＰｈｅＴＳ；４‐ニトロベンゼンスルフィン酸ナトリウム、Ｂｒ_２；（ｘｖｉｉ）Ｈ_２Ｏ_２、ＮａＩ
化合物５の合成

In some embodiments, the present invention provides a sulfurizing agent and a method for producing the same. In some embodiments, the provided sulfurizing agents were used in the synthesis of the oligonucleotides described herein. Exemplary sulfurizing agents and their synthesis are illustrated in Scheme E-1.

Scheme E-1 Sulfide synthesis

(I) MsCl, NEt ₃ ; (ii) NaMTS; (iii) PivCl, NEt ₃ ; (iv) NaMTS, NaI; (v) Compound 4; (vi) TMSCl, NEt ₃ ; (vii) Compound 35, DEAD, PPh ₃ ; (viii) TBAF; (ix) TsCl, pyridine; (x) Ac ₂ O, pyridine; (xi) KTTS; (xii) (COCl) ₂ ; (xiii) Fmoc-OSu, Py; (xiv) NaOH (Aq); (xv) Na-p-ClPheTS; Sodium 4-nitrobenzene sulphinate, Br ₂ ; (xvii) H ₂ O ₂ , NaI
Synthesis of compound 5

Compound 2: (Z) -Buta-2-ene-1,4-diol (0.93 ml, 11.3 mmol) and triethylamine (3.3 ml, 24 mmol) in dichloromethane (DCM, 50 mL), ice-cooled methane. It was added dropwise to a solution of sulfonyl chloride (1.9 ml, 24 mmol) in DCM (50 mL) with stirring. After stirring at room temperature for 0.5 hours, the mixture was poured onto ice and extracted. The organic layer was recovered, dried and _filtered . After removal of the solvent, 2.66 g of compound 2 was obtained (96%). It was determined by NMR that the compound was pure enough for direct use in the next step of the reaction. ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ5.94 (ddd, J = 5.4, 4.1, 1.3 Hz, 2H) 4.83 (dd, J = 4.1, 1.3 Hz, 4H) , 3.04 (s, 6H); ¹³ C NMR 128.34, 64.38, 38.27; MS (ESI + ve): Calculated value (M + NH ₄ ) ⁺ : 262.04, Measured value: 262.05; R _f = 0.3 (1: 1 EtOAc / Hexanes).

Compound 3: MeOH (20 ml) solution of sodium methanesulfonate (1.51 g, 11.3 mmol) in undiluted dimethanesulfonic acid (Z) -buta-2-ene-1,4-diyl (1). .25 g, 5.12 mmol) and treated at room temperature. After 5 minutes, a precipitate was observed to form. After 36 hours, the mixture was split between water and DCM. The organic layer was separated, dried (0054 ₄ ) and filtered. The solvent was removed to give a colorless oily substance. Pure compound 3 was obtained as a colorless oily substance by column chromatography (ISCO) (0.89 g, 63%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ5.84 (ddd, J = 6.6, 5.1, 1.5 Hz, 2H), 3.92 (dd, J = 5.1, 1.5 Hz, 4H) 3.33 (s, 6H); ¹³ C NMR 128.1, 51.47, 33.13; MS (ESI + ve): Calculated value (M + NH ₄ ) ⁺ : 294.00, Measured value: 294.04; R _f = 0.4 (1: 1 EtOAc / Hexanes).

Compound 4: Morpholine (10 g, 115 mmol) was added to ethylene sulfide (15 g, 250 mmol) in a round bottom flask under an argon atmosphere. The reaction was stirred for 7 hours and loaded directly into a silica gel column. The column was first washed with DCM and then using 2% MeOH / DCM to give compound 4 as a colorless oil (15.3 g, 91%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ3.67-3.59 (m, 4H), 2.63-2.52 (m, 2H), 2.51-2.45 (m, 2H), 2. 44-2-34 (m, 4H); MS (ESI + ve): Calculated value (M + H) ⁺ = 148.07, measured value: 148.1.

Compound 5: A solution of 2-morpholinoethanethiol (0.21 g, 1.44 mmol) in DCM (1 mL) into a solution of compound 3 (0.40 g, 1.44 mmol) in DCM (10 mL) at room temperature with stirring. Dropped using a syringe. Immediately after addition, the reaction was confirmed by TLC and showed rapid formation of the product and some dimers. After 0.5 hours, the mixture was split by the addition of water. Extraction was performed to separate the organic layer, dried (silyl ₄ ) and filtered. After removing the solvent under vacuum, compound 5 was obtained as a colorless oily substance by column chromatography (0.29 g, 58%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ5.78 (m, 2H), 3.92 (d, J = 7.3 Hz, 2H), 3.70 (t, J = 4.7 Hz, 4H), 3. 46 (d, J = 5.5Hz, 2H), 3.31 (s, 3H), 2.84 (dd, J = 7.8, 6.7Hz, 2H), 2.66 (dd, J = 7) 8.8, 6.7, 2H), 2.48 (t, J = 4.6Hz, 4H); ¹³ C NMR 130.35, 126.27, 66.97, 58.20, 53.67, 51. 52, 36.22, 35.16, 33.67; MS (ESI + ve): calculated value (M + H) ⁺ : 344.05, measured value: 344.06; R _f = 0.3 (EtOAc).

Compound 5b: A solution of compound 4b (395 mg, 1.085 mmol) in DCM (1 mL) was added dropwise to a solution of compound 3 (300 mg, 1.085 mmol) in DCM (15 mL) at room temperature using a syringe. .. After 1 hour, the resulting solution was divided by the addition of water. Extraction was performed to separate the organic layer, dried (silyl ₄ ) and filtered. After removing the solvent under vacuum, compound 5b was obtained as a colorless oily substance by column chromatography (0.35 g, 58%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ5.83-5.70 (m, 2H) 5.35-5.21 (dt, J = 26.0, 9.3 Hz, 2H) 5.16-5 .07 (m, 1H), 4.59-4.54 (d, J = 9.5Hz, 1H), 4.29-4.23 (m, 1H), 4.23-4.18 (m, 1H) 3.99-3.88 (dd, J = 6.7, 1.2Hz, 2H) 3.80-3.72 (ddd, J = 10.1, 4.6, 2.6Hz, 1H), 3.64-3.56 (m, 1H), 3.50-3.43 (m, 1H), 3.31 (s, 3H), 2.09 (s, 3H), 2.03 (S, 6H), 2.00 (s, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ170.68, 170.30, 169.51, 169.30, 129.43, 127.14, 87. 73, 76.49, 73.89, 69.16, 67.99, 61.99, 51.64, 35.89, 33.58, 20.95, 20.80, 20.74, 20.71; MS (ESI + ve): Calculated value (M + NH ₄ ) ⁺ : 578.07, measured value: 577.96; R _f = 0.5 (1: 1 EtOAc / hexane).

Synthesis of compound 7

Compound 6: Ice-cooled (Z) -buta-2-ene-1,4-diol (0.93 ml, 11.3 mmol) and triethylamine (1.6 mL, 11.5 mmol) in a DCM (50 ml) solution, chloride. Treatment was carried out with pivaloyl (1.4 ml, 11.4 mmol) in a dripping manner using a syringe over 2 minutes or more. After 1 hour, TLC showed good reaction results. The resulting mixture was divided by the addition of water. Extraction was performed to separate the organic layer, dried (silyl ₄ ) and concentrated under vacuum. TLC (Rf = 0.6, 1: 1 EtOAc / Hexanes) revealed that this crude compound did not contain the starting diol, and the crude compound was used to prepare the mesylate. The crude material was placed in DCM (50 ml) containing triethylamine (1.7 mL, 12 mmol) and cooled on an ice bath. Methanesulfonyl chloride (0.98 ml, 12.66 mmol) was added dropwise over 2 minutes using a syringe. Immediately after addition, TLC showed that the starting material was completely consumed. The resulting mixture was divided by the addition of water. Extraction was performed to separate the organic layer, dried (silyl ₄ ) and concentrated under vacuum. Column chromatography gave pure compound 6 as a colorless oil (1.48 g, 52%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ5.89-5.75 (m, 2H) 4.89-4.84 (d, J = 5.7 Hz, 2H) 4.68-4.63 (d) , J = 5.9Hz, 2H), 3.03 (s, 3H), 1.19 (s, 9H); ¹³ C NMR (100MHz, CDCl ₃ ) δ178.28, 130.61, 126.11, 65 .08, 59.65, 38.84, 38.21, 27.25; MS (ESI + ve): Calculated value (M + NH ₄ ) ⁺ : 268.12, Measured value: 268.20; R _f = 0.3 ( 20% EtOAc / Hexanes).

Compound 7: MeOH (10 ml) of sodium methanesulfonate (0.63 g, 4.70 mmol) and pivalic acid (Z) -4- (methylsulfonyloxy) pig-2-enyl (1.00 g, 4.00 mmol). The solution was stirred at room temperature for 18 hours to form a white precipitate (after 10 minutes). The resulting mixture was split by the addition of water and DCM. Extracted with DCM, the organic layer was separated, dried (ו ₄ ), filtered and concentrated under vacuum. Column chromatography gave compound 7 as a colorless oil (0.83 g, 78%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ5.82-5.73 (m, 2H), 4.73-4.66 (m, 2H), 3.95-3.87 (m, 2H), 3. 32 (s, 3H), 1.19 (s, 9H); ^{13 1} C NMR (100 MHz, CDCl ₃ ) δ178.35, 129.37, 127.32, 59.50, 51.44, 38.84, 33 .61, 27.28; MS (ESI + ve): Calculated (M + NH ₄ ) ⁺ : 284.10, Measured: 284.19; R _f = 0.4 (20% EtOAc / Hexanes).

Synthesis of compound 9

Compound 9: Pivaloyl chloride (0.60 g, 5.0 mmol) was added dropwise to a solution of S-2-hydroxyethyl methanesulfonate (0.65 g, 4.16 mmol) in DCM (20 ml) with stirring. After 2 hours at room temperature, the resulting mixture with a white precipitate was divided with water. The organic layer was separated, dried (Na ₂ SO ₄ ), filtered and concentrated to give an oily substance. Column chromatography gave compound 9 as a colorless oil (0.45 g, 45%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ4.39-4.34 (t, J = 6.3 Hz, 2H) 3.44-3.39 (t, J = 6.3 Hz, 2H) 3.36 (S, 3H), 1.20 (s, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ62.10, 51.11, 38.96, 35.19, 27.24; MS (ESI + ve): Calculation Value (M + NH ₄ ) ⁺ : 158.08, measured value: 158.04; R _f = 0.3 (20% EtOAc / Hexanes).

Synthesis of compound 12

Compound 11: Pivaloyl chloride (4.96 ml, 40.3 mmol) in a DCM (50 mL) solution of ice-cooled 2-hydroxymethylphenol (5 g, 40.3 mmol) and triethylamine (5.61 ml, 40.3 mmol). Dropped using a syringe. The ice-cooled crude pivalic acid ester solution was treated with triethylamine (6.74 ml, 48.4 mmol) and 50 mL of DCM. Methanesulfonyl chloride (3.43 ml, 44.3 mmol) was then added slowly using a syringe (5 minutes) and the resulting mixture was warmed to room temperature. The mixture was poured onto ice, the organic layer was separated, then washed with saturated NaHCO ₃ (aq), dried (0054 ₄ ), filtered and concentrated to give 10.5 g of a pale yellow crude oil. .. Pure compound 11 was obtained by column chromatography (ISCO) (5.45 g, 47%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ7.53-7.46 (dd, 7.7, 1.8 Hz, 1H), 7.46-7.40 (dt, 7.7, 1.8 Hz, 1H) , 7.32-7.24 (t, 7.7Hz, 1H), 7.13-7.06 (d, 7.7Hz, 1H), 5.21 (s, 2H), 2.79 (s, 3H), 1.40 (s, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ177.05, 150.06, 131.18, 131.07, 126.35, 125.94, 123.21, 66 .88, 39.48, 38.82, 27.30, 27.26; MS (ESI + ve): Calculated value (M + NH ₄ ) ⁺ : 304.12, Measured value: 303.99; R _f = 0.4 ( 20% EtOAc / Hexanes).

Compound 12: A MeOH (20 mL) solution of sodium methanesulfonate (0.825 g, 6.15 mmol) to room temperature with 2-((methylsulfonyloxy) methyl) phenyl (1.76 g, 6.15 mmol) pivalate. And stirred for 18 hours. The mixture was split between water and DCM. The organic layer was separated, dried (0054 ₄ ), filtered and concentrated to give a colorless oily substance. Column chromatography gave pure compound 12 as a thin colorless oil (0.754 g, 41%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ7.48-7.44 (dd, J = 7.7, 1.7 Hz, 1H), 7.39-7.34 (td, J = 7.8, 1. 7Hz, 1H), 7.25-7.20 (td, J = 7.6, 1.2Hz, 1H), 7.10-7.06 (dd, J = 8.2, 1.2Hz, 1H) 4.29 (s, 2H), 2.90 (s, 3H), 1.39 (s, 9H); ¹³ C NMR (100MHz, CDCl ₃ ) δ176.69, 149.59, 131.17, 129 .85, 127.41, 126.18, 123.40, 51.43, 39.47, 36.01, 27.30; MS (ESI + ve): Calculated value (M + NH ₄ ) ⁺ : 320.10. : 320.09; R _f = 0.4 (20% EtOAc / hexane).

Synthesis of compound 14

Compound 14: Chloromethyl pivalate (0.478 ml, 3.32 mmol), sodium iodide (0.050 g, 0.33 mmol) and sodium methanesulfonate (0.445 g, 3.32 mmol) in acetone (7 ml). ) Was added to the mixture at room temperature with stirring. After 24 hours, TLC showed good conversion to product. The solvent was removed and the residue was split between water and DCM. The organic layer was separated, dried (0054 ₄ ), filtered and concentrated to give a colorless oily substance. Column chromatography gave pure compound 14 as a slightly pink solid (0.41 g, 55%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ 5.67 (s, 2H) 3.39 (s, 3H), 1.24 (s, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 177.35, 67 .84, 52.20, 38.93, 27.05; R _f = 0.5 (20% EtOAc / Hexanes).

Synthesis of compound 16

Compound 16: Prepared from compound 15 and NaMTS as previously described in US Pat. No. 3,484,473. ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ 4.86 (s, 2H) 3.45 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 52.15, 41.50.

Synthesis of compounds 18 and 19

Compound 18: Prepared from Compound 17 and NaMTS as previously described: Chem. Pharm. Bull. Vol. 12 (11) p. 1271, 1964. ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ3.55 (s, 4H) 3.40 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ50.67, 35.96.

Compound 19: 2-Molholinoethanethiol (0.17 g, 1.2 mmol) in DCM (1 mL) is mixed with compound 18 (300 mg, 1.2 mmol) in DCM (10 mL) at room temperature with stirring. Dropped using. Immediately after addition, TLC showed rapid formation of the product and some dimers. After 0.5 hours, the mixture was split by the addition of NaHCO ₃ solution. Extraction was performed to separate the organic layer, dried (silyl ₄ ), filtered and concentrated under vacuum. Pure compound 19 was obtained as a colorless oily substance by column chromatography (0.20 g, 53%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ3.73-3.67 (t, J = 4.7 Hz, 4H), 3.51-3.46 (m, 2H), 3.35 (s, 3H), 3.07-3.01 (m, 2H), 2.88-2.83 (m, 2H), 2.69-2.63 (m, 2H), 2.52-2.43 (t, J) = 4.6Hz, 4H); ¹³ C NMR (100MHz, CDCl ₃ ) δ66.96, 57.91, 53.58, 50.79, 37.66, 36.10, 35.52; MS (ESI + ve) :. Calculated value (M + H) ⁺ : 318.03, measured value: 318.04; R _f = 0.3 (EtOAc).

Synthesis of compound 22

Compound 21: Compound 20 was converted to compound 21 by a procedure similar to the procedure described for compound 11. ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ7.45-7.36 (m, 4H) 5.37 (s, 2H) 5.21 (s, 2H) 2.93 (s, 3H), 1 .21 (s, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ178.20, 135.65, 131.92, 130.48, 129.98, 129.78, 128.88, 69.05, 63 .39, 38.94, 38.36, 27.27; MS (ESI + ve): Calculated value (M + NH ₄ ) ⁺ : 318.24, Measured value: 318.14; R _f = 0.4 (20% EtOAc / Hexane).

Compound 22: Compound 21 was converted to compound 22 by a procedure similar to the procedure described for compound 12. ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ7.46-7.32, (m, 4H), 5.21 (s, 2H), 4.50 (s, 2H), 3.03 (s, 3H), 1.21 (s, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ178.24, 135.10, 133.15, 130.93, 130.32, 129.05, 129.00, 63.61, 51.07, 38.97, 38.03, 27.30; MS (ESI + ve): Calculated value (M + NH ₄ ) ⁺ : 334.11, Measured value: 334.13; R _f = 0.4 (20% EtOAc) / Hexane).

Synthesis of compound 25

Compound 23: Compound 23 is prepared according to the method of the literature (Journal of Medicinal Chemistry, 50 (23), 5568-5570, 2007).

Compound 24: A solution of ice-cooled compound 23 (1 mmol) in pyridine (10 mL) is treated with acetyl chloride (1 mmol) while dropping, and then after 5 minutes, subsequently treated with MsCl (1.1 mmol) while dropping. .. Warm the solution to room temperature and then remove the solvent. The residue is dissolved in EtOAc, washed with water, dried (ו ₄ ), filtered and concentrated under vacuum. Purification by column chromatography gives pure compound 24.

Compound 25: Compound 24 is converted to compound 25 by a procedure similar to the procedure described for compound 12.

Synthesis of compound 27

Compound 27: Compound 26 was converted to compound 27 by a procedure similar to that described for compound 14. ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ 3.97 (s, 2H) 3.79 (s, 3H) 3.48 (s, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ168.84, 53 .35, 51.53, 37.83; MS (ESI + ve): Calculated (M + NH ₄ ) ⁺ : 202.02, Measured: 201.96; R _f = 0.2 (20% EtOAc / Hexanes).

Synthesis of compound 29

Compound 29: Compound 28 was converted to compound 29 by a procedure similar to that described for compound 14. ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ3.72 (s, 3H) 3.39 (t, J = 6.8 Hz, 2H) 3.34 (s, 3H) 2.85 (t, J = 6.8Hz, 2H); ¹³ C NMR (100MHz, CDCl ₃ ) δ171.53, 52.29, 50.66, 34.51, 31.20; MS (ESI + ve): Calculated value (M + NH ₄ ) ⁺ : 216 .10, measured value: 216.04; R _f = 0.2 (20% EtOAc / Hexanes).

Synthesis of compound 31

Compound 30: Compound 30 is prepared according to the method of the literature (Tetrahedron, 42 (2), 601-607; 1986).

Compound 31: Compound 31 is prepared from compound 30 according to patented procedures (US Pat. No. 20090181444).

Synthesis of compound 33

Compound 33: Compound 33 is prepared from compound 32 in accordance with patented procedures (US Pat. No. 20090181444).

Synthesis of compound 38

Compound 36: A solution of ice-cooled compound 34 (1 mmol) in DCM (20 mL) is treated with NEt ₃ (1 mmol) and then TMS-Cl (1.1 mmol) is added dropwise. After 1 hour, the solution is washed with water, dried (ו ₄ ), filtered and concentrated under vacuum. This crude TMS-protected material was redissolved in THF (10 mL), followed by PPh ₃ (1.2 mmol), compound 35 (1.2 mmol), then DEAD (1.2 mmol, dripping). Add. After stirring at room temperature for 18 hours, the solvent is removed under vacuum, the residue is redissolved in DCM, the solution is washed with water, dried (0054 ₄ ), filtered and concentrated under vacuum. Purification by column chromatography gives pure compound 36.

Compound 37: A solution of compound 36 (0.5 mmol) in THF (10 mL) is treated with TBAF (1 mmol of 1 M solution in THF) while being monitored by TLC. When TMS cleavage is complete, the solvent is removed under vacuum, the residue is redissolved in DCM, the solution is washed with water, dried (regsvr ₄ ), filtered and concentrated under vacuum. The crude alcohol is redissolved in pyridine (5 mL) and TsCl (0.55 mmol) is added. After 18 hours at room temperature, the solvent is removed, the residue is redissolved in DCM, the solution is washed with water, dried (0054 ₄ ), filtered and concentrated under vacuum. Purification by column chromatography gives compound 37.

Compound 38: Compound 37 is converted to compound 38 by a procedure similar to that described for compound 12.

Synthesis of compound 41

Compound 40: A solution of ice-cooled compound 39 (1 mmol) in DCM (20 mL) is treated with NEt ₃ (1 mmol), after which TMS-Cl (1.1 mmol) is added dropwise. After 1 hour, the solution is washed with water, dried (ו ₄ ), filtered and concentrated under vacuum. This crude TMS-protected material was redissolved in THF (10 mL), into which PPh ₃ (1.2 mmol), potassium p-toluenethiosulfonate (KTTS, 1.2 mmol), anhydrous ZnCl ₂ (1 mmol), then Continue to add DEAD (1.2 mmol, dripping) to. After stirring at room temperature for 18 hours, the solvent is removed under vacuum, the residue is redissolved in DCM, the solution is washed with water, dried (0054 ₄ ), filtered and concentrated under vacuum. Purification by column chromatography gives pure compound 40.

Compound 41: A solution of compound 40 (0.5 mmol) in THF (10 mL) is treated with TBAF (1 mmol of 1 M solution in THF) while being monitored by TLC. When TMS cleavage is complete, the solvent is removed under vacuum, the residue is redissolved in DCM, the solution is washed with water, dried (0054 ₄ ), filtered and concentrated under vacuum. The crude alcohol is redissolved in THF (10 mL), to which PPh ₃ (1.2 mmol), compound 35 (1.2 mmol) and then DEAD (1.2 mmol, instilling) are added in succession. After stirring at room temperature for 18 hours, the solvent is removed under vacuum, the residue is redissolved in DCM, the solution is washed with water, dried (0054 ₄ ), filtered and concentrated under vacuum. Purification by column chromatography gives compound 41.

Synthesis of compound 43

Compound 43: Compound 42 is converted to compound 43 by a procedure similar to that described for compound 14.

Synthesis of compounds 45a, 45b, 47a and 47b

Compound 45a: 4- (2-chloroethyl) morpholine hydrochloride (Compound 44) (50 g, 269 mmol), sodium iodide (4.03 g, 26.9 mmol) and potassium 4-methylbenzenesulfonate (73.0 g, 322 mmol). ) Was stirred in MeOH (200 ml) and heated at 60 ° C. for 72 hours. The pale yellow mixture is cooled, diluted with 200 mL of water, stirred for 0.5 hours, then the white solid is collected by filtration, water (100 mL), IPA (200 mL), EtOAc (200 mL) and ether (200 mL). ) Was washed. The dry mass was 68 g. The microcrystalline powder was recrystallized from water (200 mL) at 90 ° C., allowed to stand overnight at room temperature, and then the crystalline mass was recovered by filtration (64 g, 71%). ^{1 1} H NMR (500 MHz, CDCl ₃ ) δ7.83 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 3.67 (t, J = 4.7 Hz) 4,H), 3.16 (t, J = 6.9Hz, 2H), 2.64 (t, J = 6.9Hz, 2H), 2.47 (s, 3H), 2.41 (t, J) = 4.4Hz, 4H); ^{13 1} C NMR (100MHz, CDCl ₃ ) δ144.82, 141.99, 129.95, 127.14, 66.85, 56.54, 53.18, 33.44, 21 .78; MS (ESI + ve): Calculated value (M + H) ⁺ : 302.08, Measured value: 302.22.

Compound 45b: Compound 45b was synthesized by substituting potassium 4-methylbenzenesulfonate with potassium 4-chlorobenzenesulfonate, in a manner similar to that described for compound 45a. (ESI + ve): Calculated value (M + H) ⁺ : 324.03, 322.04, measured value: 324.22, 322.20 ( ³⁷ Cl, ³⁵ Cl isotope pattern).

Compound 47a: Replacing 4- (2-chloroethyl) morpholine hydrochloride with 4- (2-bromoethyl) -N-methylpiperazine hydrobromide (Compound 46) in a manner similar to that described for Compound 45a. Compound 47a was synthesized. MS (ESI + ve): Calculated value (M + H) ⁺ : 315.12, measured value: 315.07.

Compound 47b: Substituting 4- (2-bromoethyl) -N-methylpiperazine hydrobromide with 4- (2-bromoethyl) -N-methylpiperazine hydrobromide and 4-methyl by a method similar to that described for compound 45a. Compound 47b is synthesized by replacing potassium benzenesulfonate with potassium 4-chlorobenzenesulfonate.

Synthesis of compound 50

Compound 49: A mixture of compound 48 (500 mg, 1.894 mmol) and sodium methanesulfonate (534 mg, 3.98 mmol) was dissolved in acetone (10 ml) and stirred at room temperature for 4 hours. TLC showed a complete reaction. The solvent was removed and then the mixture was split by the addition of water and DCM. Extraction was performed to separate the organic layer, dried ( _Л4 ), filtered and concentrated under vacuum. Column chromatography gave the pure product as a colorless solid (0.60 g, 97%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ7.47 (dd, J = 5.5, 3.5 Hz, 2H), 7.38 (dd, J = 5.5, 3.5 Hz, 2H), 4.55 (S, 4H), 3.13 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ133.41, 131.75, 129.51, 51.02, 37.09; MS (ESI + ve): Calculation Value (M + NH ₄ ) ⁺ : 344.01, measured value: 344.01; R _f = 0.5 (1: 1 EtOAc / Hexanes).

Compound 50: A solution of compound 4 (180 mg, 1.225 mmol) in DCM (2 mL) was added dropwise to a solution of compound 49 (400 mg, 1.225 mmol) in DCM (20 mL) at room temperature using a syringe. .. After 0.5 hours, the mixture was split by the addition of NaHCO ₃ . Extraction was performed to separate the organic layer, which was then dried (cav ₄ ), filtered and concentrated under vacuum. The product was obtained as a colorless oily substance by column chromatography (170 mg, 35%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ7.43-7.39 (m, 1H), 7.35-7.27 (m, 3H), 4.54 (s, 2H), 4.03 (s, 2H), 3.67 (t, J = 4.6Hz, 4H), 3.05 (s, 3H), 2.58-2.50 (m, 4H), 2.38 (t, J = 4. 6Hz, 4H); ¹³ C NMR (100MHz, CDCl ₃ ) δ136.09, 133.20, 131.87, 131.21, 128.86, 128.54, 66.95, 57.95, 53.52, 51.04, 40.81, 38.18, 35.82; MS (ESI + ve): calculated value (M + H): 394.06, measured value: 394.23; R _f = 0.5 (EtOAc).

Synthesis of compound 55

Compound 54: Bromine (0.246 ml, 4.78 mmol) in DCM (50 mL), sodium 4-nitrobenzenesulfinate (2 g, 9.56 mmol) and 2-hydroxyethyl disulfide (0.585 ml) in DCM (50 mL). The mixture was added dropwise to the mixture (4.78 mmol) over 0.5 hours at room temperature with stirring. After 2 hours at room temperature, the mixture was filtered to remove salt. The solvent was removed and then column purification by column chromatography gave the product as a colorless oil (2.1 g, 83%). Rf = 0.4 (1: 1 EA / Hexane. ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ8.41 (d, J = 8.8 Hz, 2H), 8.13 (d, J = 8.8 Hz, 2H) ), 3.89 (t, J = 5.8Hz, 2H), 3.23 (t, J = 5.8Hz, 2H), 2.02-1.94 (s, br, 1H); ^{13 1} C NMR (100 MHz, CDCl ₃ ) δ149.73, 128.40, 124.82, 60.94, 39.28.

Compound 55: Compound 51 (1 g, 3.07 mmol) in DCM (50 mL) was treated with oxalyl dichloride (0.527 ml, 6.15 mmol) while dropping, and the mixture was stirred at room temperature. During the reaction, a uniform colorless liquid was formed. After 1 hour at room temperature, the solvent was removed under vacuum. The white residue (crude acid chloride, compound 52) was redissolved in DCM (20 mL) and added to a solution of compound 54 (0.48 g, 3.1 mmol) in pyridine (20 mL) with stirring. After 18 hours, it was determined that the reaction was complete. The solvent was removed. The residue was redissolved in toluene and split with water. Toluene extract was recovered, washed with saturated brine, dried ( _Л4 ), filtered and concentrated. Column chromatography gave pure compound 55 as a colorless solid (0.86 g, 60%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ8.31 (d, J = 8.9 Hz, 2H), 8.03 (d, J = 8.9 Hz, 2H), 7.7 (d, J = 7.5 Hz) , 2H), 7.58 (d, J = 7.5Hz, 2H), 7.42 (t, J = 7.5Hz, 2H), 7.32 (dt, J = 7.5, 1.2Hz, 2H), 5.22 (s, br, 1H), 4.34 (s, br, 4H), 4.19 (t, J = 6.8Hz, 1H), 3.23 (t, J = 6. 0Hz, 2H), 4.17 (s, br, 6H); ^{13 1} C NMR (100MHz, CDCl ₃ ) δ173.91, 155.10, 150.60, 149.53, 143.85, 141.42, 128 .27, 128.20, 127.91, 127.22, 127.18, 125.12, 124.84, 120.17, 66.81, 62.60, 56.40, 47.25, 34.76 , 25.37; MS (ESI + ve): calculated value (M + H) ⁺ : 571.11, measured value: 571.00; R _f = 0.5 (EtOAc).

Synthesis of compound 59

Compound 57: Compound 57 is prepared from compound 56 (1 mmol) by reacting with 1.5 equivalents of Fmoc-OSu in pyridine for 18 hours at room temperature. Separation with water is then performed by column chromatography to give pure compound 57.

Compound 59: Compound 59 is prepared from compound 57 by a procedure similar to that described for compound 55.

Synthesis of compound 63

Compound 61: Compound 61 is prepared from compound 60 by a procedure similar to that described for compound 57.

Compound 63: Compound 63 is prepared from compound 61 by a procedure similar to that described for compound 55.

Synthesis of compound 69

Compound 65: Ethyl isobutyrate (11.6 g, 100 mmol) was added to a chilled (-78 ° C.) LDA in THF solution (53 mL, 2 M) over 0.5 hours. The orange mixture was then briefly warmed to 0 ° C. and recooled to −78 ° C. Then 1,2-dibromoethane (20 g, 106 mmol) was added over 0.5 hours, the solution was slowly warmed to room temperature and stirred overnight. The resulting mixture was split by the addition of water and EA. Extracted to EA, washed with saturated brine, the orange layer was separated, dried (ו ₄ ), filtered and concentrated under vacuum. Column chromatography gave the pure product as a colorless oil (6.0 g, 25%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ4.15 (q, J = 7.1 Hz, 2H), 3.35 (t, J = 8.4 Hz, 2H), 2.16 (t, J = 8.4 Hz) , 2H), 1.27 (t, J = 7.1Hz, 3H), 1.22 (s, 6H); ¹³ C NMR (100MHz, CDCl ₃ ) δ176.78, 60.83, 43.83, 42 .89, 28.45, 25.20, 14.32; R _f = 0.5 (5% EtOAc / hexane).

Compound 66: A mixture of compound 65 (3.4 g, 15.24 mmol) and morpholine (13.28 g, 152 mmol) in THF (20 ml) was heated at 50 ° C. for 72 hours in a glass pressure vessel. The formation of a white precipitate was observed. The mixture was split between water and EA. The EA extract was dried ( _Л4 ), filtered and concentrated. The product was obtained as a colorless liquid by column chromatography (3.5 g, 100%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ4.11 (q, J = 7.1 Hz, 2H) 3.67 (t, J = 4.7 Hz, 4H) 2.41 (t, br, J = 4) .0Hz, 4H), 2.30-1.26 (m, 2H), 1.74-1.70 (m, 2H), 1.23 (t, J = 7.1Hz, 3H), 1.17 (S, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ177.69, 67.11, 55.15, 54.02, 41.10, 37.04, 25.49, 14.35; MS (ESI + ve) ): Calculated value (M + H) ⁺ : 230.18, measured value: 230.33; R _f = 0.5 (5% EtOAc / hexane).

Compound 67: Compound 66 (120 mg, 0.523 mmol) and sodium hydroxide (120 mg, 3.00 mmol) were stirred together in 1: 1 EtOH / H ₂ O (10 mL). After 36 hours, the pH of the solution was adjusted to about 2 by the addition of concentrated HCl, then NEt ₃ was added until the pH reached about 10. SiO ₂ (2 g) was added to evaporate the solvent / water. Column chromatography with dry filling (MeOH / DCM with 2% NEt ₃ in DCM) gave the product as a partial (about 1/3 mol equivalent) HNEt ₃ salt. The adjusted yield was 103 mg, 84%. ^{1 1} H NMR (399 MHz, CDCl ₃ and 3 drops of DMSO-d6) δ3.62 (t, J = 4.7 Hz, 4H), 2.51-2.46 (br, 4H), 2.40 (t, J = 7.1Hz, 2H), 1.62 (J = 7.1Hz, 2H), 1.08 (s, 6H); ¹³ C NMR (100MHz, CDCl ₃ ) δ179.91, 65.91, 54. 29, 52.87, 45.44, 41.37, 35.09, 25.93, 8.56; MS (ESI-ve): Calculated value (MH) ^- : 200.13, Measured value: 200 .16; R _f = 0.5 (2% NEt ₃ /10% MeOH / DCM).

Compound 69a: A suspension of compound 67 (11 g, 54.9 mmol) in DCM (200 mL) was treated with oxalyl chloride (9.42 ml, 110 mmol) and stirred at room temperature. During the reaction, a uniform colorless solution was formed. After 1 hour at room temperature, the solvent was removed under reduced pressure. The yellow residue is suspended in a mixture of DCM (200 mL) and Py (100 mL), followed by S-2-hydroxyethyl compound 54 (15.91 g, 60.4 mmol) (DB-7-5) all at once. In addition, the reaction was monitored by HPLC / MS. After 18 hours, MS and TLC determined that the reaction was substantially complete. The solvent was removed and then the residue was redissolved in DCM / bicarbonate. The combined organic matter was dried (ו ₄ ), filtered and concentrated. Purification by column chromatography gave 12.5 g, 51% of compound 69a as a yellow oil. This oily substance was allowed to stand and crystallized. ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ8.41 (d, J = 8.5 Hz, 2H), 8.13 (d, J = 8.5 Hz, 2H), 4.27 (t, J = 6.3 Hz) , 2H) 3.65 (t, J = 4.6Hz, 4H) 3.28 (t, J = 6.3Hz, 2H) 2.40 (s, br, 4H) 2.27 (t) , J = 7.7Hz, 2H), 1.71 (t, J = 7.7Hz, 2H), 1.14 (s, 6H); ¹³ C NMR (100MHz, CDCl ₃ ) δ177.11, 150.71 , 149.69, 128.34, 124.92, 66.96, 61.66, 54.95, 53.92, 41.25, 46.81, 35.11, 25.36; MS (ESI + ve) :. Calculated value (M + H) ^- : 447.12, measured value: 446.98.

Compound 69b: A suspension of compound 67 (128 mg, 0.636 mmol) in DCM (12 mL) was treated with oxalyl chloride (545 μl, 6.36 mmol) in a dropwise manner and stirred at room temperature. During the reaction, a uniform colorless solution was formed, which immediately precipitated a colorless precipitate. HPLC / MS showed good conversion to acid chloride (Compound 68), as shown by conversion to N-propylamide. The solvent was removed under reduced pressure. The white residue is cooled on an ice bath and treated with a solution of S-2-hydroxyethyl methanesulfonate (Compound 54) (99 mg, 0.636 mmol) in DCM (12 mL), followed by stirring with a Hunig base (0. 34 g, 2.6 mmol, 4 eq) was added dropwise. After 1.5 hours, the solution was washed with diluted NaHCO ₃ (aq). The combined extracts were dried (ו ₄ ), filtered and concentrated. Column chromatography gave the product as a colorless oil (84 mg, 39%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ4.40 (t, J = 6.2 Hz, 2H) 3.70 (t, J = 4.6 Hz, 4H) 3.44 (t, J = 6.2 Hz) , 2H), 3.39 (s, 3H), 2.47-1.43 (br, 4H), 2.32 (t, J = 7.7Hz, 2H), 1.77 (t, J = 7) .7Hz, 2H), 1.64-1.58 (br, 4H), 1.22 (s, 6H); ¹³ C NMR (100MHz, CDCl ₃ ) δ177.30, 67.08, 62.27, 55 .03, 54.01, 51.07, 41.32, 36.99, 35.14, 25.44; MS (ESI + ve): Calculated value (M + H) ⁺ : 340.13, Measured value: 340.27; R _f = 0.2 (EtOAc).

Synthesis of compound 73

Compound 70: Compound 70 was prepared by reacting N-methylpiperazine with compound 65 by a procedure similar to that described for compound 66. ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ4.10 (q, J = 7.1 Hz, 2H), 2.3-2.6 (br, 8H), 2.28 (t, J = 8.0 Hz, 2H) , 2.26 (s, 3H), 1.72 (t, J = 8.0Hz, 2H), 1.23 (t, J = 7.1Hz, 3H), 1.15 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ177.71, 60.44, 55.27, 54.64, 53.50, 46.18, 41.15, 37.36, 25.47, 14.34; MS (ESI + ve): Calculated value (M + H) ⁺ : 243.20, measured value: 243.31.

Compound 71: Compound 71 was prepared by hydrolysis of compound 70 using a procedure similar to that described for compound 67. ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ12-10 (vbr, 1H) 3.2-2.2 (vbr, 8H) 2.44 (t, J = 8.0 Hz, 2H) 2.36 ( s, 3H), 1.71 (t, J = 8.0Hz, 2H), 1.17 (s, 6H), ¹³ C NMR (100MHz, CDCl ₃ ) δ181.36, 55.18, 53.51, 52.18, 44.31, 41.57, 37.19, 26.28; MS (ESI + ve): Calculated value (M + H) ⁺ : 215.17, Measured value: 215.19.

Compound 83: Bromine (2.58 ml, 50.0 mmol) in DCM (50 mL), sodium benzenesulfinate (16.4 g, 100 mmol) and 2-hydroxyethyl disulfide (6.11 ml, 49) in DCM (50 mL). The mixture was added dropwise to the mixture (.9 mmol) over 0.5 hours at room temperature with stirring. After 2 hours at room temperature, the TLC showed a good reaction (Rf = 0.4 (1: 1 EA / hexane)), so the mixture was filtered to remove salts. The solvent was removed and then column chromatography gave the product as a colorless oil (17.4 g, 80%).

Compound 73: Compound 71 (1.342 g, 6.29 mmol) in DCM (20 mL) was treated with oxalyl chloride (1.079 ml, 12.58 mmol) and stirred at room temperature. During the reaction, the formation of a pale yellow precipitate was observed. After 0.5 hours at room temperature, the solvent was removed under reduced pressure. The pale yellow residue was suspended in a mixture of DCM (20 mL) and Py (20 mL), then compound 83 (2.060 g, 9.44 mmol) was added all at once. After 18 hours, TLC determined that the reaction was complete. The solvent was removed and then the residue was redissolved in water and washed with ether. The aqueous layer was concentrated to give a brown solid. Recrystallization from boiling EtOH (10 mL) gave an HCl salt with slight impurities. This was used as a free base (NET ₃ in DCM) and directly filled on silica gel. Column chromatography gave 0.95 g, 36% pure compound 73. ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ7.96 (d, J = 8.0 Hz, 2H), 7.68 (t, J = 7.2 Hz, 1H), 7.59 (t, J = 7.7 Hz) , 2H), 4.25 (t, J = 6.3Hz, 2H), 3.25 (t, J = 6.3Hz, 2H), 2.8-2.3 (vbr, 8H), 2.36 (S, 3H), 2.33 (t, J = 7.7Hz, 2H), 1.74 (t, J = 7.7Hz, 2H), 1.16 (s, 6H); ¹³ C NMR (100MHz) , CDCl ₃ ) δ177.13, 144.68, 134.09, 129.59, 127.09, 61.99, 54.86, 54.35, 52.84, 45.95, 45.71, 41. 26, 37.05, 34.66, 25.39; MS (ESI + ve): Calculated value (M + H) ⁺ : 415.17, Measured value: 415.09.

Synthesis of compound 76

Compound 75: Compound 74 was converted to compound 75 by a procedure similar to that described for compound 2. ^{1 1} H NMR (500 MHz, CDCl ₃ ) δ6.49-6.41 (br, d, J = 6.3 Hz, 1H), 4.91 (dt, J = 6.3, 2.7 Hz, 1H), 4 .59 (ddd, J = 31.4, 10.6, 3.0Hz, 2H), 3.84 (s, 3H), 3.04 (s, 3H), 2.09 (s, 3H); 170 .27, 169.05, 68.90, 53.33, 52.03, 37.63, 23.16; MS (ESI + ve): Calculated value (M + H) ⁺ : 240.06, Measured value: 240.24.

Compound 76: Compound 75 was converted to compound 76 by a procedure similar to that described for compound 3. ^{1 1} H NMR (500 MHz, CDCl ₃ ) δ6.56-6.40 (br, d, J = 6.2 Hz, 1H), 4.93 (q, J = 5.9 Hz, 1H), 3.81 (s) , 3H), 4 3.74 (dd, J = 14.6, 4.8Hz, 1H), 3.57 (dd, J = 14.6, 5.6Hz, 1H), 3.38 (s, 3H) ), 2.07 (s, 3H); ¹³ C NMR (126 MHz, CDCl ₃ ) δ170.44,170.19,53.23,52.02,50.73,37.77,23.12; MS ( ESI + ve): Calculated value (M + H) ⁺ : 256.03, measured value: 256.21.

Synthesis of compound 78

A mixture of compound 77 (2.49 ml, 32.8 mmol) and sodium methanesulfonate (4.4 g, 32.8 mmol) was stirred in acetone (80 ml) for 6 hours. Acetone was removed by rotary evaporation and the mixture was disrupted in DCM. After filtration, the DCM was removed by evaporation (crude yield 5 g) and the mixture was subjected to purification by column chromatography. The yield of pure compound 78 was 2.8 g, 55%, a colorless oily substance, Rf = 0.3 (20% EA / hexane). ^{1 1} H NMR (500 MHz, CDCl ₃ ) δ 5.30 (s, 2H) 3.48 (s, 3H) 3.38 (s, 3H); ¹³ C NMR (126 MHz, CDCl ₃ ) δ 80.07, 57 .40, 22.71.

Synthesis of compound 80

A solution of ice-cooled compound 79 (5 g, 65.7 mmol) and triethylamine (11 ml, 79 mmol) in DCM (150 mL) was treated with methanesulfonyl chloride (5.59 ml, 72.3 mmol) over 2 minutes. After 1 hour, TLC determined that the reaction was complete. Water was added and the organic extract was washed successively with dilute HCl, saturated sodium bicarbonate and saturated brine, then dried (ו ₄ ), filtered and concentrated. The product mesylate (9.4 g, 61 mmol, 96%) was placed in acetone (200 ml), then the potassium toluenethiosulfinate (61 mmol) was added and the solution was stirred at 50 ° C. for 18 hours. The formation of a viscous white precipitate was observed. The mixture was filtered and concentrated to an oily substance, which was then extracted into DCM / water. The organic extract was dried ( _Л4 ), filtered and concentrated. Column chromatography gave pure compound 80 as a colorless oily substance. This oily substance was allowed to stand and crystallized. Rf = 0.3 (20% EA / hexane).

Synthesis of compound 82

Of 2-chloro-N, N-dimethylethaneamine hydrochloride (10 g, 69.4 mmol), sodium iodide (1.041 g, 6.94 mmol) and potassium 4-methylbenzenesulfonate (18.86 g, 83 mmol). The mixture was stirred in MeOH (50 ml) and heated at 60 ° C. for 72 hours. Post-treatment with water and then column chromatography gave pure compound 82 as a colorless oil (8.5 g, 47%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ7.81 (d, J = 8.4 Hz, 2H), 7.33 (dd, J = 8.4, 0.6 Hz, 2H), 3.09 (t, J) = 6.8Hz, 2H), 2.53 (t, J = 6.8Hz, 2H), 2.44 (s, 3H), 2.17 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ144.76, 142.03, 129.92, 127.18, 57.51, 45.14, 34.29, 21.77; MS (ESI + ve): Calculated value (M + H) ⁺ : 260.07, measured value : 260.16.

Synthesis of compound 85

Sodium methanesulphonate (11.5 g, 86 mmol) was placed in a dry 50 mL single-mouth flask. Using a dry glass syringe, 30 mL of anhydrous DMF (Aldrich) was added. Using a dry glass syringe, 5 mL of 3-bromopropionitrile (Compound 84) (8.2 g, 61.2 mmol) was added. The reaction flask was closed under argon, sealed and stirred at 50 ° C. for 24 hours. Reactions were monitored using TLC (system: hexane / ethyl acetate-5: 5-v / v). The reaction mixture was diluted with 100 mL of ethyl acetate and washed 5 times with water (5 x 100 mL). The organic layer was dried over sodium sulfate, filtered and evaporated to dryness. The residue was dissolved in 5 mL of dichloromethane and purified by silica gel chromatography (CombiFlash) using a linear gradient of ethyl acetate in hexanes. Pure compound 85 was obtained as a colorless oil (7.2 g, 52%). ^{1 1} H-NMR (CDCl ₃ , 399 MHz) δ3.43 (s, 3H), 3.41 (t, 2H, J = 2.5 Hz), 2.93 (t, 2H, J = 2.5 Hz); ¹³ C-NMR (CDCl ₃ , 100 MHz) δ117.7, 51.2, 31.7, 19.6.

Synthesis of compound 87

Compound 87: Compound 86 (1.11 mL, 12.5 mmol) and sodium 4-nitrobenzenesulfinate (5.23 g, 25.0 mmol) were added to DCM (12.5 mL) to give a white suspension. Bromine (0.64 ml, 12.5 mmol) was added to the solution with stirring. The solution was stirred at room temperature for 30 minutes and filtered to remove salt and then the filtrate was evaporated under reduced pressure. The crude product was placed on a silica gel column and eluted with hexane- EtOAc to give pure compound 87 (4.80 g, 20.6 mmol, 82% yield). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ8.44-8.40 (m, 2H), 8.15-8.10 (m, 2H), 2.58 (s, 3H); ¹³ C NMR (100 MHz, 100 MHz, CDCl ₃ ) δ149.00, 128.54, 124.87, 94.61, 18.55; R _f = 0.60 (1: 1 EtOAc / Hexanes).

Synthesis of compound 90

Compound 89: N- (2-mercaptoethyl) acetamide (11.92 g, 100 mmol) of compound 88 was dissolved in EtOAc (300 mL) in a 1 L round bottom flask to give a colorless solution. Sodium iodide (0.150 g, 1.000 mmol) and 30% hydrogen peroxide (3.40 g, 100 mmol) in water (11.3 mL) were added to the solution and the solution was stirred at room temperature for 45 minutes. Saturated Na ₂ S ₂ O ₃ was added to the solution. The aqueous layer was extracted with EtOAc (3 x 300 mL). The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated by rotary evaporation. The crude product was subjected to column chromatography and eluted with a MeOH / DCM gradient to give pure compound 89 (4.94 g, 20.90 mmol, 41.8% yield). This compound 89 was determined by TLC to be sufficiently pure for direct use in the next step of the reaction. Rf = 0.35 (9: 1 DCM / methanol).

Compound 90: Compound 89 (2.364 g, 10.00 mmol) and sodium 4-nitrobenzenesulfinate (4.18 g, 20 mmol) are dissolved in CH ₂ Cl ₂ (20 mL) in a 100 mL round bottom flask to provide a white suspension. A turbid liquid was obtained. Bromine (0.516 ml, 10.00 mmol) was added to the solution with stirring. The solution was stirred at room temperature for 30 minutes and filtered to remove salt and then the filtrate was evaporated under reduced pressure. The crude product was placed on a silica gel column and eluted with hexane- EtOAc to give pure compound 90 (2.37 g, 7.79 mmol, 39% yield). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ8.45-8.40 (m, 2H), 8.17-8.12 (m, 2H), 3.60-3.54 (dt, 2H), 3. 21-3.16 (t, 2H), 2.00 (s, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ130.35, 126.27, 66.97, 58.20, 53.67, 51 .52, 36.22, 35.16, 33.67; R _f = 0.40 (EtOAc).

Synthesis of compounds 93 and 94

Compound 92: Sodium 4-chlorobenzenesulfonate (14.5 g, 63.0 mmol) was dissolved in MeOH in a 250 mL round bottom flask. Compound 91 cis-1,4-dichloro-2-butene (3.16 g, 30.0 mmol) and sodium iodide (450 mg, 3.0 mmol) were added to the solution with stirring. The solution was stirred at room temperature for 3 hours and then heated to 50 ° C. After stirring at 50 ° C. for 18 hours, the solvent was removed under reduced pressure. The crude material produced was diluted with DCM (300 mL) and washed with water (1 x 100 mL). The aqueous layer was extracted with DCM (1 x 100 mL). The combined organic layer was dried over Na ₂ SO ₄ , filtered and concentrated. The crude product was subjected to column chromatography and eluted with an EtOAc / Hexane gradient to give compound 92 (5.99 g, 12.8 mmol, 43% yield). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ7.87-7.84 (m, 4H), 7.56-7.51 (m, 4H), 5.56-5.50 (m, 2H), 3. 72-3.68 (m, 4H); ¹³ C NMR (100 MHz, CDCl ₃ ) 130.35, 126.27, 66.97, 58.20, 53.67, 51.52, 36.22, 35. 16, 33.67; R _f = 0.35 (1: 3 EtOAc / Hexanes).

Compound 93: Compound 92 (3.09 g, 6.58 mmol) was dissolved in THF at 0 ° C. in a 100 mL round bottom flask. Triethylamine (459 μL, 3.29 mmol) and 2-methyl-2-propanethiol (742 μL, 6.58 mmol) were added to the solution with stirring at 0 ° C. The solution was stirred at 0 ° C. for 20 minutes, then triethylamine (230 μL, 1.65 mmol) was added to the solution with stirring at 0 ° C. After stirring at 0 ° C. for 40 minutes, the solvent was removed under reduced pressure. The product was subjected to column chromatography and eluted with an EtOAc / Hexane gradient to give compound 93 (1.77 g, 4.62 mmol, 70% yield). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ7.90-7.85 (m, 2H), 7.56-7.52 (m, 2H), 5.73-5.65 (m, 1H), 5. 55-5.48 (m, 1H), 3.78-3.75 (d, 2H), 3.36-3.32 (d, 2H), 1.32 (s, 9H); ¹³ C NMR ( 100 MHz, CDCl ₃ ) δ143.54, 140.60, 130.90, 129.83, 128.66, 124.56, 48.29, 37.05, 33.35, 30.16; R _f = 0. 60 (1: 3 EtOAc / Hexanes).

Compound 94: Using a procedure similar to that described for compound 93, 2-propanethiol was used instead of 2-methyl-2-propanethiol to give pure compound 94 (1.13 g, 3.06 mmol, yield 72%). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ7.90-7.82 (m, 2H), 7.56-7.48 (m, 2H), 5.72-5.64 (m, 1H), 5. 57-5.47 (m, 1H), 3.79-3.72 (d, 2H), 3.33-1.26 (d, 2H), 3.61-2.92 (m, 1H), 1.27 (d, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ143.28, 140.36, 131.37, 125.63, 124.55, 124.56, 41.32, 38.02, 35.81, 33.08, 22.54; R _f = 0.55 (1: 3 EtOAc / Hexanes).

Synthesis of compound 96

Compound 96: Compound 9 (1.81 g, 5.50 mmol) and compound 54 (2.17 g, 8.25 mmol) were mixed and dehydrated by simultaneous evaporation with anhydrous toluene (3 × 2 mL). The mixture was dissolved in dehydrated 1,2-dichloroethane (16.5 mL). 4 Å of molecular sieves were added to the solution and the mixture was stirred at room temperature for 30 minutes. Trimethylsilyl triflate (497 μL, 2.75 mmol) was added to the solution with stirring. The solution was stirred at room temperature for 9 hours, then additional compound 54 (0.723 g, 2.75 mmol) was added to the solution with stirring. After stirring at room temperature for 16 hours, the solution was diluted with DCM (100 mL) and washed with saturated NaHCO ₃ (1 x 100 mL). The aqueous layer was extracted with DCM (1 x 50 mL). The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated by rotary evaporation. The crude product was subjected to column chromatography and eluted with an EtOAc / Hexane gradient to give compound 96 (2.21 g, 3.73 mmol, 68% yield). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ8.42-8.36 (d, 2H), 8.12-8.06 (d, 2H), 6.07-6.00 (d, 1H), 5. 33-5.30 (d, 1H), 5.23-5.15 (dd, 1H), 4.69-4.63 (d, 1H), 4.15-4.04 (m, 3H), 4.04-3.87 (m, 2H), 3.84-3.73 (m, 1H), 3.22-3.12 (t, 2H), 2.12-1.92 (m, 12H) ); ¹³ C NMR (100 MHz, CDCl ₃ ) 179.95, 170.74, 170.48, 150.77, 149.59, 128.47, 125.03, 101.40, 71.08, 70.02 , 67.39, 66.92, 61.76, 51.12, 36.51, 23.72, 20.92; R _f = 0.25 (1: 9 MeOH / DCM).

Synthesis of compound 100

Compound 98: A solution of ice-cooled compound 97 (4.87 g, 22.0 mmol) in dehydrated pyridine (70 mL) with phenoxyacetic acid anhydride (37.8 g, 132.0 mmol) and 4-dimethylaminopyridine (26.9 mg,). It was treated with 220 μmol). After stirring at room temperature for 12 hours, the mixture was concentrated under vacuum and co-evaporated with toluene. The product was diluted with DCM (400 mL) and washed with saturated NaHCO ₃ (1 x 400 mL). Extraction was performed to separate the organic layer, dried over Na ₂ SO ₄ , filtered and concentrated under vacuum. The product was subjected to column chromatography and eluted with an EtOAc / Hexane gradient to give pure compound 98 (16.7 g, 22.0 mmol, 100% yield). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ7.36-6.75 (m, 20H), 6.27-6.20 (d, 1H), 5.43-5.36 (m, 1H), 5. 22-5.06 (m, 2H), 4.81-4.65 (m, 3H), 4.60-4.52 (m, 2H), 4.45-4.29 (m, 2H), 4.17-4.02 (m, 2H), 3.97-3.87 (m, 1H), 1.85 and 1.74 (d, 3H, rotational isomers); ¹³ C NMR (100 MHz, CDCl). ₃ ) δ170.20, 168.78, 168.70, 168.36, 167.52, 157.56, 157.48, 157.40, 130.09, 129.70, 129.66, 129.60, 122.32, 122.01, 121.94, 114.61, 114.58, 114.58, 114.39, 91.93, 68.37, 68.25, 67.30, 64.54, 61. 25, 46.43, 22.95; R _f = 0.35 (1: 1 EtOAc / Hexanes).

Compound 99: Compound 98 (15.15 g, 20.0 mmol) is dissolved in ClCH ₂ CH ₂ Cl (40 mL), and trimethylsilyl trifluoromethanesulfonate (5.43 mL, 30.0 mmol) is added to the solution with stirring at room temperature. added. The solution was stirred at 50 ° C. for 24 hours, triethylamine (12.6 mL, 90.0 mmol) was added and the mixture was concentrated under vacuum. The product was purified by silica gel column chromatography (EtOAc (2.5% Et 3N) _-hexane ) to give compound 99 with a small amount of impurities, containing trace amounts of phenoxyacetic acid. This material was used in the next step of the reaction scheme without further purification.

Compound 100: Compound 99 (3.63 g, 6 mmol) and S-2-hydroxyethyl-4-nitrobenzenesulfonothioate (2.76 g, 10.5 mmol) of compound 54 are dissolved in ClCH ₂ CH ₂ Cl (18 mL). To obtain a colorless solution. 4 Å molecular sieves were added to the solution at room temperature with stirring. The solution was stirred at room temperature for 30 minutes and then trimethylsilyl trifluoromethanesulfonate (0.543 ml, 3.00 mmol) was added. The mixture was stirred at room temperature for 24 hours and TLC showed that the reaction was about 90% complete. Further compound 54 (237.0 mg, 0.90 mmol) was added to the mixture and stirred for an additional 36 hours to complete the reaction. The mixture was diluted with DCM (100 mL) and washed with saturated NaHCO ₃ (1 x 100 mL). The aqueous layer was back-extracted with CH ₂ Cl ₂ (1 x 50 mL). The combined organic layers were dried over Na ₂ SO ₄ , filtered, concentrated and dissolved in 120 mL of Ac ₂ O-pyridine (1: 9, v / v). The mixture was stirred for 12 hours and then concentrated under reduced pressure. The product was diluted with CH ₂ Cl ₂ (100 mL) and washed with saturated NaHCO ₃ (1 x 100 mL). The aqueous layer was back-extracted with CH ₂ Cl ₂ (1 x 50 mL). The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated. The product was purified by silica gel column chromatography (EtOAc-hexane gradient) to give pure compound 100 (2.56 g, 2.94 mmol, 49.0% yield). ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ8.70-8.62 (d, 2H), 8.41-8.33 (d, 2H), 7.65-7.03 (m, 15H), 6. 35-6.27 (d, 1H), 5.81-5.72 (m, 2H), 5.06-4.97 (m, 3H), 4.92-4.85 (m, 2H), 4.83-4.62 (m, 2H), 4.58-4.44 (m, 2H), 4.34-4.26 (m, 2H), 4.26-4.15 (m, 1H) ), 4.06-3.95 (m, 1H), 3.51-1.40 (m, 2H), 2.22 and 2.18 (d, 3H, rotational isomers); ¹³ C NMR (100 MHz). , CDCl ₃ ) δ171.17, 169.13, 169.05, 168.92, 157.86, 157.80, 157.76, 150.82, 149.63, 130.01, 129.96, 128. 47, 125.06, 122.27, 122.23, 114.93, 114.74, 10.086, 70.64, 70.54, 65.36, 64.90, 62.21, 51.30, 36.54, 23.74; R _f = 0.60 (1: 1 EtOAc / Hexanes).

Synthesis of compound 104

Compound 102: A procedure similar to that described for compound 98 is used to use compound 101 instead of compound 97 to obtain pure compound 102.

Compound 103: A procedure similar to that described for compound 99 is used to use compound 102 instead of compound 98 to obtain pure compound 103.

Compound 104: A procedure similar to that described for compound 100 is used to use compound 103 instead of compound 99 to obtain pure compound 103.

Synthesis of compounds 109 and 111

Compound 106: A dehydrated 1,2-dichloroethane (300 mL) solution of compound 105 (100 mmol) is treated with TiCl ₄ (110 mmol) over 2 minutes while dropping using a syringe. After reflux for 16 hours, TLC indicates completion of the reaction. The crude is diluted with DCM and washed with saturated NaHCO ₃ . The combined organic layer is dried over Na ₂ SO ₄ , filtered, concentrated and used in the next reaction without further purification.

Compound 107: Thiourea (150 mmol) and compound 106 (100 mmol) are dissolved in acetone (200 mL) under argon. The reaction mixture is heated to 60 ° C. and stirred. After 2 hours, the white precipitate is removed by filtration. Recrystallize the precipitate. Filtered crystals and Na ₂ S ₂ O ₅ (140 mmol) are added to the mixture of DCM (500 mL) and H ₂ O (250 mL) with stirring. The reaction mixture is heated to reflux under Ar. After 3 hours, the reaction mixture is cooled to room temperature and the phases are separated. The aqueous layer is back-extracted with DCM. The combined organic layer is dried over Na ₂ SO ₄ , filtered and concentrated to give compound 107.

Compound 108: 1-bromo-2-chloroethane (100 mmol), triethylamine (200 mmol), and compound 107 (100 mmol) are dissolved in acetonitrile (200 mL) under Ar. The reaction mixture is heated to 60 ° C. and stirred. TLC indicates that the reaction is complete. The mixture is diluted with DCM (500 mL) and washed with NaHCO ₃ (250 mL). The organic layer is dried over Na ₂ SO ₄ and filtered, concentrated and purified to give compound 108.

Compound 109: Compound 112 (120 mmol), sodium iodide (10.0 mmol), and compound 108 (100 mmol) are dissolved in MeOH (200 mL) under Ar. The reaction mixture is heated to 60 ° C. and stirred. TLC indicates that the reaction is complete. The mixture is diluted with DCM (500 mL) and washed with NaHCO ₃ (250 mL). The organic layer is dried over Na ₂ SO ₄ and filtered, concentrated and purified to give compound 109.

Compound 110: 1-bromo-4-chloro-2,3-cis-butene (100 mmol), triethylamine (200 mmol), and compound 107 (100 mmol) are dissolved in acetonitrile (200 mL) under Ar. The reaction mixture is heated to 60 ° C. TLC indicates that the reaction is complete. The mixture is diluted with DCM (500 mL) and washed with NaHCO ₃ (250 mL). The organic layer is dried over Na ₂ SO ₄ and filtered, concentrated and purified to give compound 110.

Compound 111: Compound 112 (120 mmol), sodium iodide (10.0 mmol), and compound 110 (100 mmol) are dissolved in MeOH (200 mL) under Ar. The reaction mixture is heated to 60 ° C. TLC indicates that the reaction is complete. The mixture is diluted with DCM (500 mL) and washed with NaHCO ₃ (250 mL). The organic layer is dried over Na ₂ SO ₄ and filtered, concentrated and purified to give compound 111.

Synthesis of compound 115

Compound 113: 2-Chlorosulfonyl acetonitrile (Compound 113) is prepared according to the Sammes procedure (as described in patent GB1252903).

Compound 114: Sodium sulfide nine hydrate (65 mmol) is placed in 100 mL of water. Hold the flask in a water bath. Compound 113 (50 mmol) is added dropwise to the solution with stirring while gently warming the water bath. In the flask, the appearance of sulfur is observed, after which the sulfur disappears. The solvent is evaporated under vacuum and the residue is recrystallized from ethanol. In this way, sodium cyanomethanesulfonate (Compound 114) is obtained as a colorless crystalline solid.

Compound 115: In a 100 mL round bottom flask, compound 114 (13.69 g, 86 mmol) is added to anhydrous DMF (30 mL) with stirring. Next, 3-bromopropionitrile (5 mL, 8.2 g, 61.2 mmol) is added and the resulting mixture is stirred at 50 ° C. for 18 hours while monitoring by TLC. When the reaction is complete (when 3-bromopropionitrile is completely consumed), the reaction mixture is diluted with EtOAc (100 mL) and the organic layer is washed with 5 x 20 mL _H2O . The separated organic layer is then dried (0054 ₄ ), filtered and concentrated by rotary evaporation to give an oily substance. The crude oily material is purified by column chromatography using an EtOAc / Hexane gradient, the fraction containing the high purity material is recovered, the solvent is removed by rotary evaporation, and then further dried under vacuum. , The pure compound 115 is obtained as a colorless oily substance.

Synthesis of compound 118

Compound 116: Compound 51 (2.9 g, 8.91 mmol) in DCM (50 mL) was treated with oxalyl dichloride (0.53 ml, 6.15 mmol) while dropping, and then added dropwise with DMF (10 μL). The treatment was carried out while stirring at room temperature. During the reaction, a uniform colorless solution was formed. After 2 hours at room temperature, the solvent was removed under reduced pressure. The white residue was redissolved in DCM (20 mL) and then added dropwise to a solution of ethane-1,2-dithiol (8.40 g, 89 mmol) in pyridine (10 mL) with UPLC / MS. After 18 hours, it was determined that the reaction was complete. The solvent was removed and the residual ethanethiol was removed by distillation from trap to trap, then the residue was purified by column chromatography with DCM to give pure compound 116 as a colorless solid. The yield was 1.82 g, 51%. MS (ESI +): Calculated value (M + Na) ⁺ : 424.10, Measured value: 424.06; ^{1 1} H NMR (500 MHz, CDCl ₃ ) δ7.79 (d, J = 7.5 Hz, 2H), 7.63 (D, J = 5.7Hz, 2H), 7.43 (t, J = 7.5Hz, 2H), 7.34 (t, J = 7.4Hz, 2H), 5.40-5.15 ( br, 1H), 4.60-4.35 (br, 2H), 4.31-4.19 (m, 1H), 3.15-3.04 (br, 2H), 2.95-2. 80 (m, 1H), 2.75-2.60 (br, 2H), 1.72-1.43 (m, 6H); ¹³ C NMR (126 MHz, CDCl ₃ ) δ203.14, 154.75, 143.92, 141.49, 127.84, 127.19, 125.17, 120.13, 66.81, 62.69, 47.41, 33.01, 25.78, 24.60.

Compound 117: Sodium iodide (6.35 mg, 0.042 mmol) and hydrogen peroxide (0.144 g, 4.23 mmol) (30%) in a solution of compound 116 (1.7 g, 4.23 mmol) in EtOAc (12 mL). 0.45 mL of aqueous solution) was added and the mixture was stirred at room temperature for 15 minutes. TLC showed that the starting material was completely consumed. Hydrous sodium thiosulfate was added until the solution became colorless. The solution was washed with water, dried ( _Л4 ) and then column chromatographically (EtOAc / Hexanes) to give pure compound 117 as a colorless solid foam (1.45 g, 86%). MS (ESI +): Calculated value (M + H) ⁺ : 82.07, Measured value: 82.08; ^{1 1} H NMR (500MHz, CDCl ₃ ) δ7.78 (d, J = 7.6Hz, 4H), 7.63 (D, J = 5.8Hz, 4H), 7.42 (t, J = 7.5Hz, 4H), 7.33 (t, J = 7.5Hz, 4H), 5.42-5.25 ( br, 2H), 4.55-4.35 (br, 4H), 4.30-4.18 (br, 2H), 3.25-2.10 (br, 4H), 2.95-2. 70 (br, 4H), 1.65-1.45 (br, 12H); ¹³ C NMR (126 MHz, CDCl ₃ ) δ203.19, 154.71, 143.90, 141.45, 127.80, 127 .17, 125.16, 120.10, 66.76, 62.65, 47.37, 37.90, 28.56, 25.73.

Compound 118: Bromine (0.091 ml, 1.77 mmol), a mixture of sodium 4-nitrobenzenesulfinate (0.742 g, 3.55 mmol) and compound 117 (1.42 g, 1.773 mmol) in DCM (10 mL). The mixture was added dropwise at room temperature over 2 minutes with stirring. After stirring at room temperature for 30 minutes, the mixture was filtered and the filtrate was concentrated under vacuum to give an orange solid foam. Column chromatography (DCM / hexane) gave pure compound 118 as a pale yellow solid foam (1.26 g, 60%). MS (ESI +): Calculated value (M + H) ⁺ : 587.71, measured value: 802.108; ^{1 1} H NMR (500 MHz, CDCl ₃ ) δ8.36 (d, J = 8.2 Hz, 2H), 8.14 (D, J = 7.3Hz, 2H), 7.79 (d, J = 7.0Hz, 2H), 7.63 (s, 2H), 7.43 (t, J = 6.7Hz, 2H) , 7.35 (d, J = 6.8Hz, 2H), 5.35-5.15 (br, 1H), 4.55-4.35 (br, 2H), 4.30-4.20 ( br, 1H), 3.30-1.00 (br, 4H), 1.70-1.25 (br, 6H); ¹³ C NMR (126 MHz, CDCl ₃ ) δ202.71, 154.66, 150. 60, 149.63, 143.78, 141.46, 128.40, 127.91, 127.21, 125.12, 124.81, 120.18, 66.86, 62.59, 47.35, 35.84, 28.42, 25.61.

Synthesis of compound 120

Compound 120: Commercially available compound 119 (1 g, 7.04 mmol) and potassium 4-methylbenzenesulfonate (1.753 g, 7.75 mmol) were stirred in MeOH (10 mL) at room temperature for 5 days, then 40. The mixture was stirred at ° C for 24 hours. TLC showed good conversion to product. The MeOH was evaporated, the residue was placed in DCM (30 mL) and Boc ₂ O (1.54 g, 7.04 mmol) was added. The mixture was treated with triethylamine (1.030 ml, 7.39 mmol) at room temperature for 10 minutes in drops until the solution was substantially uniform. It was washed with water (50 mL), dried (ו ₄ ), filtered and evaporated to give a crude product, which was further purified by column chromatography to give pure compound 120 as a colorless solid (1). .64 g, 65%). MS (ESI +): Calculated value (M + Na) ⁺ : 380.10, Measured value: 380.11; ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ7.80 (d, J = 8.2 Hz, 2H), 7.34 (Dd, J = 8.5, 0.8Hz, 2H) 5.54 (ddt, J = 24.7, 16.6, 7.9Hz, 2H) 4.65 (s, 1H), 3. 77-3.66 (m, 4H), 2.45 (s, 3H), 1.43 (s, 9H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ155.83, 145.05, 142.06, 131.80, 130.01, 127.15, 124.55, 79.72, 37.29, 32.75, 28.51, 21.79.

Synthesis of compound 122

Compound 122: A commercially available solution of compound 121 (1 g, 3.59 mmol) in water (1 mL) is treated with a solution of potassium hexafluorophosphate (0.662 g, 3.59 mmol) in water (10 mL) all at once at room temperature. The mixture was stirred by shaking for 5 minutes. The resulting solid was collected by filtration, washed with 2 x 3 mL of water and dried under vacuum with KOH to give pure compound 122 as a colorless solid (1.01 g, 82%). ^{1 1} H NMR (300 MHz, CD ₃ CN) δ3.67-3.60 (m, 2H), 3.49 (s, 3H), 3.53-3.45 (m, 2H), 3.10 (s) , 9H); ¹³ C NMR (126 MHz, CD ₃ CN) δ65.98, 54.05 (q, J = 4.1 Hz) 51.11., 28.99; ¹⁹ F NMR (376 MHz, CD ₃ CN) δ -73.15 (d, J = 706.5 Hz); ³¹ 1 P NMR (162 MHz, CD ₃ CN) δ-143.50 (hept, J = 706.6 Hz).

Synthesis of compound 125

Compound 124: A solution of commercially available compound 123 (10 mmol) in DMF (50 mL) is subsequently treated with tert-butyl-2-aminoacetate (22 mmol), diisopropylethylamine (50 mmol) and HBTU (24 mmol). After stirring at room temperature for 1 hour, water (100 mL) was added, the resulting solution was extracted with EtOAc (2 x 50 mL), washed with 5% NaHCO ₃ (2 x 25 mL), and then diluted saturated saline (2). Wash with x25 mL), dry ( _Л4 ), filter and concentrate by rotary evaporation. Purification by column chromatography gives pure compound 124.

Compound 125: Bromine (0.091 ml, 1.77 mmol), sodium 4-nitrobenzenesulfinate (0.742 g, 3.55 mmol) in DCM (10 mL) and di-tert-butyl-2,2'-((((10 mL)). A mixture of 4,4'-disulfandyl-bis (butanoyl)) bis (azandyl)) diacetate (824 mg, 1.77 mmol) is added dropwise at room temperature over 2 minutes with stirring. After stirring at room temperature for 30 minutes, the mixture is filtered and the filtrate is concentrated under vacuum to give a solid foam. Column chromatography gives the pure product as a solid foam.

Synthesis of compound 129

Compound 127: A TFA (10 mL) solution of ethane-1,2-dithiol (1.130 g, 12 mmol) was treated all at once with commercially available compound 126 (0.89 g, 9.99 mmol). The solution became warm. After stirring at room temperature for 1 hour, the volatiles were removed and the residue was subjected to column chromatography to give pure compound 127 as a colorless oil (0.95 g, 48%). R _f = 0.6 (5% MeOH / DCM); ^{1 1} H NMR (500 MHz, CDCl ₃ ) δ6.09-5.93 (br, 1H), 4.41 (d, J = 6.4 Hz, 2H) 2.87-2.83 (m, 2H), 2.79 (ddd, J = 9.7, 7.3, 3.5Hz, 2H), 2.04 (s, 3H), 1.71 ( t, J = 8.0 Hz, 1H); ¹³ C NMR (126 MHz, CDCl ₃ ) δ170.25, 40.89, 35.22, 24.91, 23.44.

Compound 129: A warm (35 ° C.) commercially available solution of compound 128 (939 mg, 3.0 mmol) in DMF (10 mL) is added dropwise with a solution of compound 127 (250 mg, 1.513 mmol) in DCM (5 mL) over 5 minutes. Processed while. After confirmation by TLC, the solvent was removed by rotary evaporation under high vacuum, then the residue was crushed with DCM (50 mL), filtered, the filtrate washed with 5 × 5% NaHCO ₃ and then dried ( ₄ ), the mixture was filtered and concentrated to obtain a pale yellow solid. Column chromatography gave pure compound 129 as a pale yellow solid. MS (ESI +): Calculated value (M + H) ⁺ : 320.02, Measured value: 320.34; ^{1 1} H NMR (500 MHz, CDCl ₃ ) δ9.28 (dd, J = 2.7, 0.7 Hz, 1H) , 8.43 (dd, J = 8.9, 2.6Hz, 1H), 7.93 (dd, J = 8.9, 0.7Hz, 1H), 6.10-5.90 (br, 1H). ), 4.41 (d, J = 6.5Hz, 2H), 3.13 (dd, J = 8.6, 6.3Hz, 2H), 2.93 (dd, J = 8.6, 6. 4Hz, 2H), 2.00 (s, 3H); ¹³ C NMR (126MHz, CDCl ₃ ) δ170.18, 168.57, 145.22, 142.22, 131.81, 119.59, 40.65 , 38.59, 30.07, 23.39.

Synthesis of compound 134

Compound 130: Compound 130 is prepared by the method previously described (Heterocycles, Vol 54, p 139, 2001).

Compound 132: Aqueous formaldehyde solution containing K ₂ CO ₃ using a procedure similar to the procedure previously described for the synthesis of compound 126 (Organic Syntheses, Coll. Vol. 6, p. 5 (1988)). Compound 131 (Tetrahedron Letters, 48 (39), 7038-7041; 2007) is converted to compound 132 by gentle heating in the presence.

Compound 133: Using a procedure similar to that described for the synthesis of compound 127, using compound 132 in place of compound 126 and compound 130 in place of ethane-1,2-dithiol. In this way, pure compound 133 is obtained.

Compound 134: Using a procedure similar to that described for the synthesis of compound 129, compound 133 is used instead of compound 127 to thus obtain pure compound 134.

Synthesis of compound 140

Compound 136: An ice-cooled solution of commercially available compound 135 (50 g, 342 mmol) and methanol (277 mL) are treated with sulfuric acid (3.36 g, 34.2 mmol) in a dropwise manner over 10 minutes and then overnight. I slowly warmed it to room temperature. Most of the solvent was removed by rotary evaporation, then saturated aqueous NaHCO ₃ solution was added carefully. The pH of the solution was adjusted to 1.9 by adding 1 M HCl, then extracted with EtOAc (200 mL), washed with diluted saturated saline (50 mL), dried ( _Л4 ) and filtered. , 29 g of colorless oily substance was obtained. Column chromatography gave pure compound 136 as a colorless solid (12.5 g, 23%). ^{1 1} H NMR (500 MHz, CDCl ₃ ) δ12.5-10.5 (vbr, 1H) 3.69 (s, 3H) 2.63 (s, 2H), 1.32 (s, 6H); ¹³ C NMR (126 MHz, CDCl ₃ ) δ183.32, 171.76, 51.72, 43.88, 40.60, 25.34.

Compound 138: Compound 136 (10 mmol) in DCM (50 mL) is treated with oxalyl dichloride (10 mmol) in a drop form, then treated with DMF (10 μL) in a drop rate, and stirred at room temperature. During the reaction, a uniform colorless solution is formed. After 2 hours at room temperature, the solvent is removed under reduced pressure. The white residue is redissolved in DCM (20 mL) and then added dropwise to a solution of compound 137 (10 mmol) in pyridine (10 mL) with stirring, monitored by TLC. After 18 hours, the solvent is removed and the residue is purified by column chromatography with DCM to give pure compound 137.

Compound 139: A solution of compound 138 (5 mmol) in THF (20 mL) is added to a solution of ice-cooled LiBH ₄ (5 mmol) in THF (20 mL). TLC is used to determine the progress of the reaction at room temperature. After the reaction is complete (after the starting material is completely consumed), water (100 mL) is carefully added to the ice-cooled solution and the solution is subsequently extracted with EtOAc (2 x 100 mL). The combined organic extracts are dried (ו ₄ ), filtered and the solvent removed by rotary evaporation. The residue is subjected to column chromatography in this way to give pure compound 139.

Compound 140: Using a procedure similar to that described for the synthesis of compound 80, compound 139 is used instead of compound 79 to thus obtain pure compound 140.

Synthesis of compound 143

Compound 139: Using a procedure similar to the procedure described for the synthesis of compound 139, compound 136 is used instead of compound 138 to obtain pure compound 141 in this way.

Compound 142: A DCM / MeOH (12/3 mL) solution of compound 141 (8.39 mmol) in an ether solution of 2 M (diazomethyl) trimethylsilane (1.05 g, 9.23 mmol) at 0 ° C. over 30 minutes. Process while dripping. After deactivating excess reagent with AcOH, wash with water (2 x 5 mL), dry (ו ₄ ), filter, remove solvent by rotary evaporation, then pure compound by column chromatography. Get 142.

Compound 143: Using a procedure similar to that described for the synthesis of compound 80, compound 142 is used instead of compound 79 to obtain pure compound 143 in this way.

Synthesis of compound 147

Compound 144: Using a procedure similar to that described for the synthesis of compound 69a, using compound 136 in place of compound 67 and using compound 54 in place of NH ₄ Cl and ⁱ Pr ₂ Net. In this way, pure compound 144 is obtained.

Compound 145: LiAlH ₄ (100 mL of 2M THF solution) is added dropwise to an ice-cooled compound 144 (86 mmol) in THF (500 mL). The mixture is warmed to room temperature and then refluxed for 0.5 hours. Residual LiAlH ₄ is disrupted by careful addition of saturated Na ₂ SO ₄ to the ice-cooled solution until a granular precipitate is formed. The mixture is filtered, concentrated and then redissolved in EtOAc, dried ( _Л4 ), filtered and concentrated under vacuum to give compound 145. This compound 145 is pure enough for direct use in the next step of the reaction.

Compound 146: Using compound 145 instead of compound 123 and Leu-Fmoc-OH instead of 2-aminoacetate tert-butyl, using a procedure similar to the procedure described for the synthesis of compound 124. Thus, pure compound 146 is obtained.

Compound 147: Using compound 146 instead of compound 79 and compound 114 instead of potassium 4-methylbenzenesulfonate, using a procedure similar to the procedure described for the synthesis of compound 80. In this way, pure compound 147 is obtained.

Synthesis of compound 150

Compound 148: Commercially available chloromethyl pivalate (10 mmol) is added dropwise to a solution of compound 130 (20 mmol) in 1,2-dichloroethane (100 mL) with stirring under argon. Heat the resulting solution for forced completion (TLC confirms the disappearance of chloromethyl pivalate). The solvent is removed by rotary evaporation and then the residue is subjected to column chromatography to give pure compound 148.

Compound 149: Using a procedure similar to that described for the synthesis of compound 89, compound 148 is used instead of compound 88 to thus obtain pure compound 149.

Compound 150: Using a procedure similar to that described for the synthesis of compound 87, compound 149 is used instead of compound 86 to thus obtain pure compound 150.

式中、Ｓ^ａは以下の構造のいずれかを表わす：

Additional Sulfide:

In the equation, ^Sa represents one of the following structures:

Synthesis of Phosphoramide In some embodiments, the present invention provides phosphoramide and a method for producing the same. In some embodiments, the provided phosphoramidite was used in the synthesis of the oligonucleotides described herein. Exemplary phosphoramides and their synthesis are described below.

Synthesis of compound 203

Compound 202: N4-Bz-5'-O-DMTr-2'-O-MOE- ⁵ -methylcitidine (Compound 201) (2.00 g, 2.77 mmol) while monitoring with TLC and UPLC / MS. Treated with 2M ammonia and pyridine (22.5 mL) in 2-propanol (Aldrich, anhydrous, 45 mL) at 60 ° C. for 5 hours and then at room temperature overnight. The solvent was removed (azeotropic mixture with 3 × toluene) and then column chromatography (0-10% MeOH / DCM) gave pure compound 202 as a colorless solid (1.62 g, 95%). From TLC and HPLC, this compound 202 was uniform. (ESI +): Calculated value (M + H) ⁺ : 618.28, measured value: 618.53; ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ7.82 (d, J = 1.3 Hz, 1H), 7.47- 7.40 (m, 2H), 7.37-7.18 (m, 8H), 6.83 (dd, J = 8.9, 1.7Hz, 4H), 5.93 (d, J = 1) .1Hz, 1H), 4.43 (td, J = 8.3, 4.9Hz, 1H), 4.24 (ddd, J = 11.7, 5.1, 3.0Hz, 1H), 4. 07 (dt, J = 8.6, 2.4Hz, 1H), 4.03-3.98 (m, 1H), 3.90 (ddd, J = 11.7, 6.3, 3.3Hz, 1H), 3.79 (d, J = 1.0Hz, 6H), 3.63-3.52 (m, 3H), 3.44 (dd, J = 11.0, 3.0Hz, 1H), 3.37 (s, 3H), 3.32 (d, J = 9.4Hz, 1H), 1.80-1.64 (s, br, 1H), 1.13 (s, 3H).

Compound 203: Isobutyric acid anhydride (0.48 ml, 2.9 mmol) was added dropwise to a solution of compound 202 (1.61 g, 2.61 mmol) in DMF (13 ml) at room temperature. The solution was stirred at room temperature for 24 hours and then the solvent was removed under vacuum at 35 ° C. Extraction to ether / bicarbonate was then dried (ו ₄ ), filtered and the solvent removed to give the crude product as a colorless solid foam. Column chromatography (0-4% MeOH / DCM) gave the pure product as a colorless solid foam (1.75 g, 98%). From TLC and HPLC, this product was uniform. (ESI +): Calculated value (M + H) ⁺ : 688.32, measured value: 688.56; ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ8.00-7.75 (br, 1H), 7.45-7. 39 (m, 2H), 7.34-7.20 (m, 8H), 6.84 (dd, J = 9.0, 1.2Hz, 4H), 5.97 (s, 1H), 4. 43 (d, J = 6.1Hz, 1H), 4.20-4.04 (m, 3H), 3.79 (d, J = 0.9Hz, 6H), 3.64-3.58 (m) , 1H), 3.64-3.52 (m, 2H), 3.47-3.40 (m, 2H), 3.38 (s, 3H), 1.70-1.55 (br, 1H). ), 1.39 (d, J = 1.0Hz, 3H), 1.18 (d, J = 6.9Hz, 6H).

Synthesis of compound 205

Compound 205: (R) -1-phenyl-1-((S) -pyrrolidin-2-yl) ethanol (Compound 4) was dried by azeotropic distillation with toluene (3 x 3 mL). A solution of dried compound 4 (0.725 g, 3.79 mmol) and 4-methylmorpholine (0.833 ml, 7.58 mmol) in toluene (5 mL) was ice-cooled with phosphorus trichloride (0.331 ml, 3.79 mmol). Was added to a solution of toluene (5 mL), which was then warmed to room temperature for 1 hour, then filtered under Ar and concentrated to give an oily substance. This oil was used in the next step of the reaction without further purification.

Synthesis of compound 207

Compound 207: Compound 207 was obtained as a crude brown oily substance using compound 206 instead of compound 204 using a procedure similar to the procedure described for the synthesis of compound 205. This oil was used in the next step of the reaction without further purification.

Synthesis of compound 208

Compound 208: Compound 203 (1.74 g, 2.53 mmol) was dehydrated by simultaneous evaporation of pyridine (3 x 5 mL) and then toluene (5 x 5 mL). The dehydrated compound 203 produced was dissolved in THF (15 mL), then triethylamine (2.47 ml, 17.7 mmol) was added and the solution was cooled to −78 ° C. by CO ₂ / acetone cooling bath method. A solution of crude compound 205 (3.79 mmol) in THF (15 mL) was added dropwise over 0.5 hours and then slowly warmed to room temperature. After 1 hour at room temperature, TLC showed that compound 203 was completely converted to product. The mixture was washed with chloroform (100 mL), placed in a separatory funnel and then extracted with NaHCO ₃ (saturated, aqueous solution, 50 mL then 2 x 25 mL). At each extraction, the aqueous extract was washed with an additional 1 x 10 mL chloroform. The combined chloroform extracts were dried (ו ₄ ), filtered and concentrated by rotary evaporation at 32 ° C. A crude solid is thus obtained, redissolved in a DCM containing a few drops of triethylamine and then subjected to column chromatography on a hexane / EtOAc gradient containing a constant concentration of 2% triethylamine to a pure compound. 208 was obtained as a white solid foam material. ^{1 1} H NMR (399 MHz, CD ₃ CN) δ7.92-7.80 (s, 1H), 7.52-7.47 (m, 2H), 7.40-7.24 (m, 12H), 6 .88 (dd, J = 9.0, 1.1Hz, 4H), 5.94 (d, J = 3.9Hz, 1H), 4.79 (dt, J = 9.6, 5.6Hz, 1H) ), 4.31-4.27 (m, 1H), 4.27-14.22 (m, 1H), 3.87 (t, J = 4.7Hz, 2H), 3.75 (d, J) = 2.0Hz, 6H), 3.66 (td, J = 5.9, 5.5, 2.2Hz, 1H), 3.58-3.53 (m, 2H), 3.50 (dd, dd, J = 11.1, 2.4Hz, 1H) 3.38 (dd, J = 11.0, 3.3Hz, 1H), 3.31 (s, 3H), 2.85 (dq, J = 10) .5, 7.4, 6.9Hz, 2H), 1.73 (s, 3H), 1.52 (d, J = 1.0Hz, 3H), 1.47-1.32 (m, 2H) , 1.17 (dd, J = 6.9, 0.7Hz, 6H), 1.05-0.90 (m, 2H); ³¹ 1 P NMR (162 MHz, CD ₃ CN) δ155.35.

Synthesis of compound 210

Compound 210: A pure compound 210 is a colorless solid using a procedure similar to that described for compound 208, using compound 209 in place of compound 203 and compound 207 in place of compound 205. Obtained as a foamy substance. ^{1 1} H NMR (399 MHz, CD ₃ CN) δ8.31-8.27 (m, 2H), 7.86 (d, J = 1.2Hz, 1H), 7.62-7.55 (m, 1H) , 7.55-7.45 (m, 5H), 7.44-7.24 (m, 12H), 6.94-6.90 (m, 4H), 5.96 (d, J = 3. 7Hz, 1H), 4.86-4.76 (m, 1H), 4.29 (dd, J = 5.0, 3.8Hz, 1H), 4.23 (dt, J = 5.8, 2) .8Hz, 1H), 3.92-3.80 (m, 2H), 3.78 (s, 6H), 3.74 (ddd, J = 7.1, 5.3, 2.1Hz, 1H) , 3.55 (t, J = 4.7Hz, 2H), 3.51 (d, J = 2.3Hz, 1H), 3.41 (dd, J = 11.1, 3.4Hz, 1H), 3.30 (s, 3H), 3.26 (ddd, J = 10.3, 6.1, 2.2Hz, 1H), 2.86 (ddt, J = 10.1, 8.2, 7. 2Hz, 1H), 1.72 (d, J = 0.7Hz, 3H), 1.62 (d, J = 1.1Hz, 3H), 1.56-1.41 (m, 2H), 1. 09-0.87 (m, 2H); ³¹ 1 P NMR (162 MHz, CD ₃ CN) δ155.22.

Synthesis of compound 212

Compound 212: Using a procedure similar to that described for compound 208, compound 211 was used instead of compound 203 to give pure compound 212 as a colorless solid foam. ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ9.25-8.70 (br, 1H), 7.69 (d, J = 1.3 Hz, 1H), 7.46-7.40 (m, 2H), 7.40-7.17 (m, 12H), 6.81 (dd, J = 9.0, 2.5Hz, 4H), 6.09 (d, J = 4.3Hz, 1H), 4.78 (Dt, J = 9.6, 5.2Hz, 1H) 4.36-4.26 (m, 2H), 3.94-3.88 (m, 2H), 3.74 (dd, J = 4.0, 0.9Hz, 6H), 3.69 (td, J = 6.4, 2.9Hz, 1H), 3.64-3.56 (m, 3H), 3.44-3.37 (M, 2H) 3.36 (d, J = 0.9Hz, 3H), 3.00 (dtd, J = 10.3, 7.9, 6.4Hz, 1H), 1.72 (s, 3H), 1.54-1.44 (m, 1H), 1.38 (dd, J = 6.8, 5.4Hz, 1H), 1.34 (d, J = 1.2Hz, 3H), 1.23-1.13 (m, 1H), 0.97-0.87 (m, 1H); ³¹ 1 P NMR (162 MHz, CDCl ₃ ) δ158.18.

Synthesis of compound 213

（ｉ）ＴＭＳＣｌ、ＮＥｔ_３、０℃；(ii)２，４，６‐トリメチルベンゼン‐１－スルホニルクロリド、ＤＭＡＰ；（ｉｉｉ）１‐メチルピロリジン、３‐ヒドロキシプロパンニトリル、ＤＢＵ、０℃；（ｉｖ）ＭｅＯＨ／Ｈ_２Ｏ、４ｄ、室温 Compound 213: Using a procedure similar to that described for compound 210, compound 211 was used instead of compound 209 to give pure compound 213 as a colorless solid foam material. ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ9.55-9.10 (br, 1H), 8.09 (d, J = 1.5 Hz, 1H), 7.80-771 (m, 2H), 7.69-7.47 (m, 12H), 7.20-7.08 (m, 4H), 6.32 (d, J = 2.9Hz, 1H), 5.20-5.11 (m) , 1H) 4.66-4.59 (m, 1H) 4.49-4.39 (m, 1H) 4.33-4.24 (m, 1H) 4.16-4.02 (M, 8H) 4.00-3.93 (m, 1H), 3.91-3.84 (m, 2H), 3.73 (dd, J = 11.0, 2.5Hz, 1H) 3.65-3.52 (m, 4H), 3.27-1.16 (m, 1H), 2.15 (s, 3H), 1.88-1.76 (m, 1H), 1 .76-1.65 (m, 1H), 1.63-1.49 (m, 4H), 1.29-1.18 (m, 1H); ³¹ 1 P NMR (162 MHz, CDCl ₃ ) δ160.08 ..

Synthesis of compound 215

(I) TMSCl, NEt ₃ , 0 ° C; (ii) 2,4,6-trimethylbenzene-1-sulfonyl chloride, DMAP; (iii) 1-methylpyrrolidine, 3-hydroxypropanenitrile, DBU, 0 ° C; (ii) iv) MeOH / H _2O , 4d, room temperature

Compound 215: (i) Temporary TMS protection : ^N2 -isobutyryl-5'-O-DMTr-2'-O-MOE-guanosine (Compound 214) (15.04 g, 21.1 mmol) and triethylamine (11. A solution of 8 ml, 85 mmol) was placed in ACN (200 mL), then chlorotrimethylsilane (10.4 ml, 82 mmol) was added dropwise to the ice-cooled solution, and the solution was stirred at room temperature for 1 hour while monitoring with TLC. .. Filtration was performed to remove the solvent, then extraction (DCM / NaHCO ₃ 500 mL / 200 mL), drying (0054 ₄ ), filtration and removal of the solvent to give the crude compound. This compound was used in the next step of the reaction without further purification. From TLC (R _f = 0.6, (5% MeOH / DCM)), the compound was uniform. (Ii) Activation of O6 site ^for cyanoethyl protection : The residue is redissolved in DCM (500 mL), then triethylamine (13.1 ml, 94 mmol) is added, followed by 2,4,6-trimethylbenzene-1. -Sulfonyl chloride (6.15 g, 28.1 mmol) and DMAP (0.14 g, 1.146 mmol) were added. The mixture was stirred at room temperature for 3 hours, monitored by TLC, until the starting material completely disappeared and new spots appeared at TLC (R _f = 0.9, (5% MeOH / DCM)). (Iii) Cyanoethyl protection : 1-methylpyrrolidine (22.43 ml, 211 mmol) is slowly added dropwise to an ice-cooled solution and TLC (new spot, R _f = 0.2, streak, (5% MeOH / DCM)). The reaction was further carried out at 0 ° C. for 1 hour while monitoring with. To the still ice-cooled solution, 3-hydroxypropanenitrile (14.40 ml, 211.0 mmol) was added dropwise, followed by DBU (6.32 mL, 42.1 mmol) and TLC (new spot, R _f) . = 0.6, (5% MeOH / DCM)) while stirring was continued for an additional 90 minutes. An additional 1.6 mL of DBU was added dropwise, carefully monitored by TLC, to force the reaction to near completion. The mixture is poured into NaH ₂ PO ₄ (50 g in 400 mL of water), the organic matter is separated, washed with diluted NaH ₂ PO ₄ (10 g in 200 mL of water), dried (ו ₄ ), filtered and rotary evaporator. Concentrated by ration. (Iv) TMS deprotection : The residue thus obtained in the previous step was redissolved in MeOH (250 mL) and then water was added dropwise, being careful not to cause precipitation. The drip was continued for 4 days while being monitored by TLC. Most of the solvent was removed, the residue was extracted (DCM / NaHCO ₃ (500/200 mL)), filtered and concentrated. The residue was subjected to column purification (0-5% MeOH / DCM) to give pure compound 215 as a white foam. From TLC and HPLC, this foam was uniform. (ESI +): Calculated value (M + H) ⁺ : 767.34, Measured value: 767.03; ¹ H NMR (300 MHz, CD ₃ CN) δ8.63 (s, 1H), 8.07 (s, 1H), 7.44-7.36 (m, 2H), 7.33-7.18 (m, 7H), 6.84-6.72 (m, 4H), 6.02 (d, J = 3.2Hz) , 1H), 4.74 (td, J = 6.2, 0.9Hz, 2H), 4.71-4.64 (m, 2H), 4.14-4.06 (m, 1H), 3 .88-3.73 (m, 8H), 3.57-3.41 (m, 4H), 3.32-3.23 (m, 3H), 3.08 (t, J = 6.2Hz, 2H) 2.77 (dt, J = 13.7, 6.9 Hz, 1H), 1.14 (dd, J = 6.9, 1.2 Hz, 6H).

Synthesis of compound 216

Compound 216: Using a procedure similar to that described for compound 208, compound 215 was used instead of compound 203 to give pure compound 216 as a colorless solid foam. ^{1 1} H NMR (399 MHz, CD ₃ CN) δ8.41 (s, 1H), 8.12 (s, 1H), 7.50-7.44 (m, 2H), 7.40-7.19 (m) , 12H), 6.80 (dd, J = 8.9, 5.2Hz, 4H), 6.04 (d, J = 5.7Hz, 1H), 4.99 (t, J = 5.5Hz, 1H), 4.94 (ddd, J = 9.9, 5.2, 3.7Hz, 1H), 4.75 (t, J = 6.2Hz, 2H), 4.29 (q, J = 3) 8.8Hz, 1H), 3.89-3.82 (m, 1H), 3.76-3.68 (m, 7H), 3.64 (ddd, J = 6.8, 5.5, 2. 3Hz, 1H), 3.50-3.46 (m, 2H), 3.42 (s, 2H), 3.38-3.28 (m, 1H), 3.21 (s, 3H), 3 .09 (t, J = 6.2Hz, 2H), 2.84 (dq, J = 10.2, 7.3Hz, 1H), 2.70-2.58 (m, 1H), 1.76 ( s, 3H), 1.46-1.27 (m, 2H), 1.12 (d, J = 6.8Hz, 3H), 1.07 (d, J = 6.8Hz, 3H), 1. 04-0.89 (m, 2H); ³¹ 1 P NMR (162 MHz, CD ₃ CN) δ154.40.

Synthesis of compound 217

Compound 217: Using a procedure similar to that described for compound 210, compound 215 was used instead of compound 209 to give pure compound 217 as a colorless solid foam. ^{1 1} H NMR (399 MHz, CD ₃ CN) δ8.56 (s, 1H), 8.13 (s, 1H), 7.44 (dd, J = 8.2, 1.5Hz, 2H), 7.39 -7.19 (m, 12H), 6.81 (dd, J = 10.4, 8.9Hz, 4H), 6.06 (d, J = 4.5Hz, 1H), 4.96-4. 86 (m, 2H), 4.75 (t, J = 6.2Hz, 2H), 4.30 (td, J = 5.1, 4.7, 2.4Hz, 1H), 3.83 (dt) , J = 11.2, 4.3Hz, 1H), 3.79-3.70 (m, 7H), 3.67 (ddd, J = 7.1, 5.2, 2.0Hz, 1H), 3.52-3.45 (m, 3H), 3.41 (dd, J = 10.8, 2.7Hz, 1H), 3.34-3.24 (m, 1H), 3.21 (s) , 3H), 3.08 (t, J = 6.1Hz, 2H), 2.89-2.72 (m, 2H), 1.68 (s, 3H), 1.54-1.38 (m) , 2H), 1.14 (dd, J = 6.8, 5.4Hz, 6H), 1.09-1.00 (m, 1H), 0.95-0.83 (m, 1H); ³¹ 1 NMR (162 MHz, CD ₃ CN) δ154.93.

Synthesis of compound 219

Compound 219: Using a procedure similar to that described for compound 208, compound 218 was used instead of compound 203 to give pure compound 219 as a colorless solid foam. ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ9.31 (s, 1H), 8.72 (s, 1H), 8.26 (s, 1H), 8.07-7.98 (m, 2H), 7 .60-7.53 (m, 1H), 7.52-7.43 (m, 4H), 7.38-7.15 (m, 12H), 6.83-6.74 (m, 4H) , 6.24 (d, J = 5.2Hz, 1H), 5.03-4.92 (m, 2H), 4.45 (q, J = 3.8Hz, 1H), 3.93 (dt, J = 11.4, 4.2Hz, 1H) 3.77 (ddd, J = 7.5, 5.8, 3.2Hz, 2H), 3.73 (s, 6H), 3.60-3 .49 (m, 3H), 3.47-1.37 (m, 2H), 3.26 (s, 3H), 3.02-2.92 (m, 1H), 1.81 (s, 3H) ), 1.56-1.43 (m, 1H), 1.37 (dq, J = 13.2, 6.5Hz, 1H), 1.22-1.09 (m, 1H), 0.96. (Ddt, J = 13.1, 7.9, 6.8 Hz, 1H); ³¹ 1 P NMR (162 MHz, CDCl ₃ ) δ157.73.

Synthesis of compound 220

Compound 220: Using a procedure similar to that described for compound 210, compound 218 was used instead of compound 209 to give pure compound 220 as a colorless solid foam material. ^{1 1} H NMR (399 MHz, CDCl ₃ ) δ9.26 (s, 1H), 8.73 (s, 1H), 8.31 (s, 1H), 8.02 (d, J = 7.6Hz, 2H) , 7.60-7.53 (m, 1H), 7.52-7.40 (m, 4H), 7.39-7.13 (m, 12H), 6.84-6.76 (m, 4H), 6.24 (d, J = 4.5Hz, 1H), 4.99 (dt, J = 10.0, 5.0Hz, 1H), 4.84 (t, J = 4.7Hz, 1H) ), 4.46 (q, J = 4.0Hz, 1H), 3.91 (dt, J = 11.1, 4.2Hz, 1H), 3.85-3.69 (m, 8H), 3. .63-3.50 (m, 3H), 3.42 (dd, J = 10.7, 4.0Hz, 1H), 3.40-3.31 (m, 1H), 3.27 (s, 3H), 3.00-2.89 (m, 1H), 1.82 (s, 3H), 1.58-1.38 (m, 2H), 1.20-1.09 (m, 1H) , 1.00 (ddt, J = 12.3, 8.8, 5.9 Hz, 1H); ³¹ 1 P NMR (162 MHz, CDCl ₃ ) δ158.98.

Synthesis of compound 222

Compound 222: Using a procedure similar to that described for compound 208, compound 221 was used instead of compound 203 to give pure compound 222 as a colorless solid foam. ^{1 1} H NMR (499 MHz, CDCl ₃ ) δ10.46 (s, br, 1H), 8.62 (d, J = 7.5 Hz, 1H), 7.42 (d, J = 7.1 Hz, 2H), 7.36-7.21 (m, 12H), 7.00 (d, J = 7.5Hz, 1H), 6.82 (dd, J = 9.0, 7.1Hz, 4H), 6.03 (S, 1H) 4.77 (td, J = 8.7, 4.8Hz, 1H), 4.34 (dt, J = 9.4, 2.3Hz, 1H), 3.97 (d, J = 4.8Hz, 1H), 3.78 (dd, J = 6.7, 3.4Hz, 1H), 3.74 (s, 3H), 3.71 (s, 6H), 3.68- 3.58 (m, 2H), 3.48-3.38 (m, 1H), 3.01 (p, J = 7.8Hz, 1H), 2.27 (s, 3H), 1.72 ( s, 3H), 1.49 (tt, J = 12.3, 7.0Hz, 1H), 1.40 (dq, J = 13.2, 6.6Hz, 1H), 1.18 (dq, J) = 13.9, 7.0 Hz, 1H), 1.02-0.90 (m, 1H); ³¹ 1 P NMR (202 MHz, CDCl ₃ ) δ158.71.

Synthesis of compound 223

Compound 223: Using a procedure similar to that described for compound 210, compound 221 was used instead of compound 209 to give pure compound 223 as a colorless solid foam. ^{1 1} H NMR (500MHz, CDCl ₃ ) δ9.76 (s, 1H), 8.70 (d, J = 7.4Hz, 1H), 7.48-7.41 (m, 2H), 7.41- 7.18 (m, 12H), 7.06 (d, J = 7.4Hz, 1H), 6.87 (dd, J = 8.9, 1.9Hz, 4H), 6.00 (s, 1H) ) 4.80 (td, J = 9.2, 4.7Hz, 1H), 4.33 (d, J = 9.5Hz, 1H), 3.84 (d, J = 4.7Hz, 1H) 3.81 (s, 3H), 3.80 (s, 3H), 3.72 (td, J = 6.6, 3.4Hz, 1H), 3.70-1.62 (m, 4H) , 3.53 (dd, J = 11.3, 2.2Hz, 1H), 3.34 (tdd, J = 10.7, 7.8, 4.9Hz, 1H), 2.96 (dq, J) = 10.3, 7.6 Hz, 1H), 2.25 (s, 3H), 1.83 (s, 3H), 1.56-1.48 (m, 1H), 1.43 (dq, J) = 13.1, 6.4 Hz, 1H), 1.24-1.16 (m, 1H), 1.00-0.91 (m, 1H); ³¹ 1 P NMR (202 MHz, CDCl ₃ ) δ157.17 ..

Synthesis of compound 225

Compound 225: Using a procedure similar to that described for compound 208, compound 224 was used instead of compound 203 to give pure compound 225 as a colorless solid foam. ^{1 1} H NMR (500 MHz, CDCl ₃ ) δ9.76 (s, br, 1H), 8.70 (d, J = 7.4 Hz, 1H), 7.48-741 (m, 2H), 7. 41-7.18 (m, 12H), 7.06 (d, J = 7.4Hz, 1H), 6.87 (dd, J = 8.9, 1.9Hz, 4H), 6.00 (s) , 1H), 4.80 (td, J = 9.2, 4.7Hz, 1H), 4.33 (d, J = 9.5Hz, 1H), 3.84 (d, J = 4.7Hz, 1H), 3.81 (s, 3H), 3.80 (s, 3H), 3.72 (td, J = 6.6, 3.4Hz, 1H), 3.69-3.62 (m, 4H), 3.53 (dd, J = 11.3, 2.2Hz, 1H), 3.34 (tdd, J = 10.7, 7.8, 4.9Hz, 1H), 2.96 (dq) , J = 10.3, 7.6Hz, 1H) 2.25 (s, 3H), 1.83 (s, 3H), 1.56-1.48 (m, 1H), 1.43 (dq) , J = 13.1, 6.4Hz, 1H), 1.24-1.16 (m, 1H), 1.00-0.91 (m, 1H); ³¹ 1 P NMR (202MHz, CDCl ₃ ) δ158 .97.

Synthesis of compound 226

Compound 226: Using a procedure similar to that described for compound 210, compound 224 was used in place of compound 209 to give pure compound 226 as a colorless solid foam. ^{1 1} H NMR (499 MHz, CDCl ₃ ) δ9.8-9.0 (br, 1H), 8.09 (d, J = 8.2 Hz, 1H), 7.40 (d, J = 7.4 Hz, 2H) ), 7.36-7.19 (m, 12H), 6.80 (dd, J = 11.4, 8.8Hz, 4H), 6.02 (d, J = 2.2Hz, 1H), 5 .22 (d, J = 8.1Hz, 1H), 4.82 (td, J = 8.1, 4.9Hz, 1H), 4.27 (d, J = 7.4Hz, 1H), 3. 96 (dd, J = 5.0, 2.2Hz, 1H), 3.81-3.76 (m, 1H), 3.74 (s, 3H), 3.70 (s, 3H), 3. 62 (s, 3H), 3.61-3.54 (m, 2H), 3.50-3.41 (m, 1H), 3.04 (dtd, J = 10.2, 8.0, 6) .3Hz, 1H), 1.75 (s, 3H), 1.56-1.47 (m, 1H), 1.44-1.35 (m, 1H), 1.26-1.18 (m) , 1H), 0.95 (dq, J = 12.3, 7.5Hz, 1H); ³¹ 1 P NMR (202MHz, CDCl ₃ ) δ158.96.

Synthesis of compound 228

Compound 228: Using a procedure similar to that described for compound 208, compound 227 was used instead of compound 203 to give pure compound 228 as a colorless solid foam. ³¹ P NMR (202 MHz, CDCl ₃ ) δ158.16.

Synthesis of compound 229

Compound 229: Using a procedure similar to that described for compound 210, compound 227 was used instead of compound 209 to give pure compound 229 as a colorless solid foam. ³¹ P NMR (202 MHz, CDCl ₃ ) δ158.84.

Synthesis of compound 231

Compound 231: Using a procedure similar to that described for compound 208, compound 230 was used instead of compound 203 to give pure compound 231 as a colorless solid foam. ^{1 1} H NMR (499 MHz, CDCl ₃ ) δ9.6-9.3 (s, 1H), 8.73 (s, 1H), 8.32 (s, 1H), 7.50-741 (m, 2H), 7.39-7.16 (m, 14H), 7.10-7.01 (m, 3H), 6.82 (d, J = 8.8Hz, 4H), 6.21 (d, J = 4.1Hz, 1H), 4.96 (dt, J = 10.1, 5.1Hz, 1H), 4.86 (s, 2H), 4.54 (t, J = 4.5Hz, 1H) ), 4.43 (q, J = 3.9Hz, 1H), 3.81-3.75 (m, 7H), 3.62 (dd, J = 10.8, 3.3Hz, 1H), 3. .54 (s, 3H), 3.46-3.42 (m, 1H), 3.41-3.33 (m, 1H), 3.01-2.93 (m, 1H), 1.82 (S, 3H), 1.56-1.47 (m, 1H), 1.44 (dq, J = 13.2, 6.5Hz, 1H), 1.22-1.11 (m, 1H) , 1.03-0.94 (m, 1H); ³¹ 1 P NMR (202 MHz, CDCl ₃ ) δ158.11.

Synthesis of compound 232

Compound 232: Using a procedure similar to that described for compound 210, compound 230 was used instead of compound 209 to give pure compound 232 as a colorless solid foam material. ^{1 1} H NMR (499 MHz, CDCl ₃ ) δ9.8-9.2 (br, 1H), 8.71 (s, 1H), 8.23 (s, 1H), 7.49-7.43 (m, 2H), 7.40-7.15 (m, 14H), 7.09-7.02 (m, 3H), 6.79 (d, J = 8.9Hz, 4H), 6.20 (d, J = 5.6Hz, 1H), 4.93 (ddd, J = 9.3, 5.0, 3.8Hz, 1H), 4.86 (s, 2H), 4.73 (t, J = 5) .3Hz, 1H), 4.43 (q, J = 3.9Hz, 1H) 3.79-3.75 (m, 1H), 3.74 (s, 3H), 3.74 (s, 3H) ), 3.57 (dt, J = 6.8, 3.8 Hz, 1H), 3.52 (s, 3H), 3.46-3.36 (m, 2H), 2.99 (dtd, J) = 10.1, 7.9, 6.5Hz, 1H) 1.80 (s, 3H), 1.54-1.45 (m, 1H), 1.39-1.31 (m, 1H) , 1.23-1.13 (m, 1H), 0.99-0.90 (m, 1H); ³¹ 1 P NMR (202 MHz, CDCl ₃ ) δ158.13.

Synthesis of compound 234

Compound 234: Using a procedure similar to that described for compound 208, compound 233 was used instead of compound 203 to give pure compound 234 as a colorless solid foam material.

Synthesis of compound 235

Compound 235: Using a procedure similar to that described for compound 210, compound 233 was used instead of compound 209 to give pure compound 235 as a colorless solid foam. ³¹ P NMR (202 MHz, CDCl ₃ ) δ158.75.

Synthesis of compound 237

Compound 237: Using a procedure similar to that described for compound 208, compound 236 was used instead of compound 203 to give pure compound 237 as a colorless solid foam material. ³¹ P NMR (202 MHz, chloroform _{-d) δ159.51} (d, JPF = 9.5 Hz).

Synthesis of compound 238

Compound 238: Using a procedure similar to that described for compound 210, compound 236 was used instead of compound 209 to give pure compound 238 as a colorless solid foam material. ³¹ P NMR (202 MHz, chloroform-d) δ159.48.

Synthesis of compound 240

Compound 240: Using a procedure similar to that described for compound 208, compound 239 was used instead of compound 203 to give pure compound 240 as a colorless solid foam material. ³¹ _P NMR (202 MHz, CDCl ₃ ) δ160.20 (d, JPF = 11.0 Hz).

Synthesis of compound 241

Compound 241: Using a procedure similar to that described for compound 210, compound 239 was used instead of compound 209 to give pure compound 241 as a colorless solid foam. ³¹ _P NMR (202 MHz, CDCl ₃ ) δ159.66 (d, JPF = 8.4 Hz).

Synthesis of compound 243

Compound 243: Using a procedure similar to that described for compound 208, compound 242 was used instead of compound 203 to give pure compound 243 as a colorless solid foam. ³¹ P NMR (202 MHz, CDCl ₃ ) δ160.20 (d, J = 11.0 Hz).

Synthesis of compound 244

Compound 244: Using a procedure similar to that described for compound 210, compound 242 was used in place of compound 209 to give pure compound 244 as a colorless solid foam. ³¹ _P NMR (202 MHz, CDCl ₃ ) δ156.52 (d, JPF = 8.5 Hz).

Synthesis of compound 246

Compound 246: Using a procedure similar to that described for compound 208, compound 245 is used instead of compound 203 to obtain pure compound 246 as a colorless solid foam material.

Synthesis of compound 247

Compound 247: Using a procedure similar to that described for compound 210, compound 245 is used instead of compound 209 to give pure compound 247 as a colorless solid foam material.

Synthesis of compound 249

Compound 249: Using a procedure similar to that described for compound 208, compound 248 is used instead of compound 203 to give pure compound 249 as a colorless solid foam material.

Synthesis of compound 250

Compound 250: Using a procedure similar to that described for compound 210, compound 248 is used instead of compound 209 to give pure compound 250 as a colorless solid foam material.

Synthesis of compound 252

Compound 252: Using a procedure similar to that described for compound 208, compound 251 is used instead of compound 203 to give pure compound 252 as a colorless solid foam material.

Synthesis of compound 253

Compound 253: Using a procedure similar to that described for compound 210, compound 251 is used in place of compound 209 to give pure compound 253 as a colorless solid foam.

Synthesis of compound 255

Compound 255: Using a procedure similar to that described for compound 208, compound 254 is used instead of compound 203 to give pure compound 255 as a colorless solid foam.

Synthesis of compound 256

Compound 256: Using a procedure similar to that described for compound 210, compound 254 is used instead of compound 209 to give pure compound 256 as a colorless solid foam.

Synthesis of compound 258

Compound 258: Using a procedure similar to that described for compound 208, compound 257 is used instead of compound 203 to obtain pure compound 258 as a colorless solid foam material.

Synthesis of compound 259

Compound 259: Using a procedure similar to that described for compound 210, compound 257 is used instead of compound 209 to give pure compound 259 as a colorless solid foam.

Example 2-16
The synthetic procedure of Example 2-16 in the DNA / RNA synthesizer ABI-394 is summarized in Table E-1 below.

Table E-1 Outline of oligonucleotide synthesis in the DNA / RNA synthesizer ABI-394 used for the synthesis of Example 2-16.

Example 2-9: Oligonucleotides containing sterically defined phosphorothioate diester nucleotide linkages according to the cycle summarized in Table E-1 were bound to a 10 μmol synthetic column and CPG with succinyl to 6.5 μmol of dC. Was synthesized on the ABI-394 DNA / RNA synthesizer. The synthesis cycle was carried out with the terminal 5'-O-DMTr group removed (DMT Off). The solid support was washed with dehydrated ACN and dried under a stream of argon. The dried solid carrier was then mixed with 5 mL of an anhydrous 1M solution of 1,8-diazabicycloundec-7-ene (DBU) in dehydrated ACN-trimethylsilyl chloride-16: 1 (v / v) at room temperature 10 Processed for minutes. Meanwhile, the solution was slowly extruded through the column with a plastic luer syringe fixed to the column outlet. The carrier was then washed with dehydrated ACN and dried under vacuum. The dried CPG was placed in a plastic vial and then treated with 3 mL of 28% aqueous ammonia for 18 hours at room temperature. The solvent was evaporated to dryness, the residue was resuspended in 10% aqueous DMSO solution and the solid support was filtered off. The crude product was purified by anion exchange preparative HPLC (0.25 to 1.75 M gradient of NaCl in 20 mM NaOH). Fractions with a purity greater than 95% were collected, concentrated and desalted by reverse phase HPLC (0-80% gradient of ACN). The final desalting product was lyophilized from water.

Examples 10-16: According to the cycle summarized in Table E-1, a 1 μmol synthetic column containing sterically defined phosphorothioate diester nucleotide linkages and 3 μmol dC succinyl bound to CPG were used. Then, it was synthesized by the ABI-394 DNA / RNA synthesizer. The synthesis cycle was carried out with the terminal 5'-O-DMTr group removed (DMT Off). The solid support was washed with dehydrated ACN and dried under a stream of argon. The dried solid carrier was then dispensed with 5 mL of an anhydrous 1M solution of 1,5-diazabicyclo (4.3.0) nona-5-ene (DBN) in dehydrated ACN-trimethylsilyl chloride-16: 1 (v / v). , Treated at room temperature for 10 minutes. The DBN solution was slowly extruded through the column with a plastic luer syringe fixed at the column outlet. The carrier was then washed with dehydrated ACN and dried under vacuum. The dried CPG was placed in a plastic vial and then treated with 2 mL of 28% aqueous ammonia for 18 hours at room temperature. The solvent was evaporated to dryness, the residue was resuspended in 10% aqueous DMSO solution and the solid support was filtered off.

Purification and desalting of Examples 2-16 The crude product was purified by a Waters 2525 BGM HPLC system equipped with a 2487 2 wavelength detector and FCO and Flex injection. An AP-1 glass column (10 × 200 mm) manufactured by Waters was filled with Source15Q Support (GE Healthcare, product number 17-0947-01) and used at a flow rate of 4 mL / min. All during purification, the column was heated using a TL600 mobile phase heater set at 75 ° C. and a TL150 temperature controller (Timberline Instruments). Buffer A: 20 mM NaOH and buffer B: 20 mM NaOH, 2.5 M NaCl were used in a graded gradient starting from 30% B to 70% B. Fractions with a purity greater than 95% are collected, concentrated and by a reverse phase column (XBride Semi Prep, 250 × 10 mm, C ₁₈ , 5 μm) with a gradient of 80% ACN from water and a flow rate of 4 mL / min. , Desalted with the same HPLC system. The final desalting product was concentrated with Specvac and then lyophilized from water.

ＬＣＭＳ分析法：
溶出剤Ａ：１５ｍＭ ＴＥＡ、４００ｍＭ ＨＦＩＰ、水
溶出剤Ｂ：５０：５０ 緩衝液Ａ／メタノール
カラム：ＵＰＬＣ＠ＯＳＴ Ｃ_１８ １．７μｍ、２．１×５００ｍｍ
カラム温度＝５０℃
勾配法：

HPLC analysis of purified oligonucleotides: The properties of the oligonucleotides were determined using DNA Pac100 (10 x 250 mm) using the following conditions:
Buffer A: 10 mM Tris HCl, 50% HCl, pH = 8.0
Buffer B: A + 0.8M NaClO ₄ , pH = 8.0
Column temperature: 60 ° C
Gradient method:

LCMS analysis method:
Eluent A: 15 mM TEA, 400 mM HFIP, water Eluent B: 50:50 Buffer A / Methanol Column: UPLC @ OST C ₁₈ 1.7 μm, 2.1 × 500 mm
Column temperature = 50 ° C
Gradient method:

Example 2 Synthesis of Oligonucleotide 101 (All- (Rp) -d [GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsCsC])

Oligonucleotide 101 was synthesized as described above. RT in IEX-HPLC: 14.70 minutes. UPLC / ESI-MS: C ₁₉₁ H ₂₄₆ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6310.2; measured value: 6310.4.

Example 3 Synthesis of oligonucleotide 102 (All- (Sp) -d [GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsCsC])

Oligonucleotide 102 was synthesized as described above. RT in IEX-HPLC: 15.49 minutes. UPLC / ESI-MS: C ₁₉₁ H ₂₄₆ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6310.2; measured value: 6310.2.

Example 4 Oligonucleotide 103 ((Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp) ] (5R-9S-5R)) Synthesis Oligonucleotide 103 was synthesized as described above. RT in IEX-HPLC: 15.10 minutes. UPLC / ESI-MS: C ₁₉₁ H ₂₄₆ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6310.2; measured value: 6310.3.

Example 5 Oligonucleotide 104 ((Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp) -d [GsCsCsTsCsAsGsTsCsCsCsCsTs ] (5S-9R-5S))

Oligonucleotide 104 was synthesized as described above. RT: 15.04 min on IEX-HPLC. UPLC / ESI-MS: C ₁₉₁ H ₂₄₆ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6310.2; measured value: 6307.2.

Example 6 Oligonucleotide 105 ((Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp) ] (1S-17R-1S))

Oligonucleotide 105 was synthesized as described above. RT in IEX-HPLC: 14.75 minutes. UPLC / ESI-MS: C ₁₉₁ H ₂₄₆ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6310.2; measured value: 6310.2.

Example 7 Oligonucleotide 106 ((Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp) -d [GsCsCsTsCsAsGsTsCsCsCsCsCsCs ] (1R-17S-1R))

Oligonucleotide 106 was synthesized as described above. RT in IEX-HPLC: 15.43 minutes. UPLC / ESI-MS: C ₁₉₁ H ₂₄₆ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6310.2; measured value: 6309.6.

Example 8 Oligonucleotide 107 ((Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp) ] ((R / S) ₉ R)) synthesis

Oligonucleotide 107 was synthesized as described above. RT: 15.02 minutes on IEX-HPLC. UPLC / ESI-MS: C ₁₉₁ H ₂₄₆ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6310.2; measured value: 6310.7.

Example 9 Oligonucleotide 108 ((Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp) -d [GsCsCsTsCsAsGsTsCsCsCsCsTs ] ((S / R) ₉ S))

Oligonucleotide 108 was synthesized as described above. RT in IEX-HPLC: 15.10 minutes. UPLC / ESI-MS: C ₁₉₁ H ₂₄₆ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6310.2; measured value: 6307.9.

Example 10 Oligonucleotide 109 ((Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp) d [GsCsCsTsCsAsGsTsCsTsCsCsTs (3S-13R-3S)) synthesis

Oligonucleotide 109 was synthesized as described above. RT in IEX-HPLC: 14.91 minutes. UPLC / ESI-MS: C ₁₉₁ H ₂₄₆ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6310.2; measured value: 6309.5.

Example 11 Oligonucleotide 110 ((Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp) -d [GsCsCsTsCsAsGsTsCsCsCsCsCsTs ] (3R-13S-3R))

Oligonucleotide 110 was synthesized as described above. RT in IEX-HPLC: 15.24 minutes. UPLC / ESI-MS: C ₁₉₁ H ₂₄₆ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6310.2; measured value: 6309.3.

Example 12 Oligonucleotide 111 ((Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp) -d [GsCsCsTsCsAsGsTsCsCsCsCsCsCs ] ((18S / R ¹⁹ ))

Oligonucleotide 111 was synthesized as described above. RT in IEX-HPLC: 15.69 minutes. UPLC / ESI-MS: C ₁₉₁ H ₂₄₆ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6310.2; measured value: 6309.4.

Example 13 Oligonucleotide 113 ((Sp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp) -d [GsCsCsTsCsAsGsTsCsCsCsCsCsCs ] (18S / R ² ))

Oligonucleotide 113 was synthesized as described above. RT in IEX-HPLC: 15.72 minutes. UPLC / ESI-MS: C ₁₉₁ H ₂₄₆ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6310.2; measured value: 6311.0.

Example 14 Oligonucleotide 114 ((Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp) -d [GsCsCsTsCsAsGsTsCsCsCsCsTs ] ((RRS) ₆ -R)) synthesis

Oligonucleotide 114 was synthesized as described above. RT in IEX-HPLC: 14.14 minutes. UPLC / ESI-MS: C ₁₉₁ H ₂₄₆ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6310.2; measured value: 6313.

Example 15 Oligonucleotide 115 ((Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp) -d [GsCsCsTsCsAsGsTsCsCsCsCsTs ] (S- (RRS) ₆ ))

Oligonucleotide 115 was synthesized as described above. RT in IEX-HPLC: 14.30 minutes. UPLC / ESI-MS: C ₁₉₁ H ₂₄₆ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6310.2; measured value: 6313.

Example 16 Oligonucleotide 116 ((Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp) d [GsCsCsTsCsAsGsTsCsTsCsCsTs Synthesis of (RS- (RRS) ₅ -RR))

Oligonucleotide 116 was synthesized as described above. RT in IEX-HPLC: 14.17 minutes. UPLC / ESI-MS: C ₁₉₁ H ₂₄₆ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6310.2; measured value: 6312.4.

The results of Examples 2-16 are summarized in Table E-2 below:

Table E-2 Outline of Example 2-16

Example 17 Synthesis of control oligonucleotide

Control oligonucleotides (see Table E-3) were synthesized using standard chemical methods of automated solid phase oligonucleotide synthesis (Beaucage, SL and Iyer, RP, Tetrahedron, 1992, 48, 2223-2311). .. More specifically, standard DNA phosphoramidite (ChemGenes Co.), ethylthiotetrazole as an activator (ETT, Muang et al., Tetrahedron Lett., 2004, 45, 6497-6499) (Glen Research), and as a sulfurizing agent. Synthesize sterically random DNA using N, N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazole-5-yl) methaneimideamide (DDTT, AM Chemicals) (Guzaev, A .; Tetrahedron Lett., 2011, 52, 434-437). The phosphoramidite coupling time was 2 minutes and the sulfurization time was 10 minutes. Oligonucleotides were deprotected and purified using standard methods. DNA phosphate diesters were synthesized using standard DNA phosphoramidite, ETT as activator, and iodine / pyridine / water as oxidant. In-house prepared 2'-O-methoxyethyl (MOE) phosphoramidite (Martin, P .; Helv. Chim. Acta. 1995, 78, 486-504; Ross, B .; Song, Q .; 2004 US Patent Gazette No. 200482775), 2'-O-methoxyethyl (MOE) DNA was synthesized using ETT as the activator and DDTT as the sulfide agent. The coupling time was 10 minutes and the sulfurization time was 10 minutes. RNA was synthesized using standard RNA phosphoramidite (ChemGenes Co.), ETT as the activator, and iodine / pyridine / water as the oxidant. The coupling time was 10 minutes.

All oligonucleotides were deprotected and purified using standard methods.

Purification of RNA (oligonucleotide 117):
Waters2525 2487 2 wavelength detector with BGM, FCO and Flex injector Buffer A: 20 mM Sodium Phosphate pH = 8.5
Buffer B: 20 mM sodium phosphate, 1 M NaBr pH = 8.5
Column: AP-1 glass column manufactured by Waters filled with Super Q-5PW (20) manufactured by Tosoh (TOSOH) and TSK Gel (anion exchange), 10 × 200 mm.
Column temperature: 70 ° C. (Timberline Instruments, TL600 mobile phase heater and TL150 temperature controller)
Gradient used:

As shown in FIG. 1, the chiral-controlled phosphorothioate diester 20-mer oligonucleotide (All- (Rp), oligonucleotide 101, FIG. 1, A) is a sterically random standard oligonucleotide of the phosphorothioate diester 20-mer. It has a different retention time than (oligonucleotide 118, FIG. 1, C) and has a sharper peak. Most of the All- (Rp) oligonucleotides (101, present in about ^1/219 fraction of the sterically random oligonucleotide 108 mixture) are probably lost during purification of the sterically random oligonucleotide 108. Those who are in the market understand that it will be.

The results of Example 17 are summarized in Table E-3 below.

Table E-3 Outline of Example 17

Procedure of Example 18-21: Oligonucleotides containing sterically defined morpholinoethyl phosphorothioate triester nucleotide linkages were oxalylated to a 1 μmol synthetic column and HCP according to the cycle summarized in Table E-4. It was synthesized with an ABI-394 DNA / RNA synthesizer using 0.8 μmol of dC. The synthesis cycle was carried out with the terminal 5'-O-DMTr group removed (DMT Off). The solid support was washed with dehydrated ACN and dried under a stream of argon. The dried HCP was placed in a plastic vial and treated with 1 mL of dehydrated propylamine (ratio 1: 4) in dehydrated pyridine for 18 hours at room temperature. The solvent was then evaporated and the residue was resuspended in a 10% DMSO-containing aqueous solution with a pH of about 1.5 and the HCP carrier was filtered off. The crude product was purified by reverse phase preparative HPLC. Fractions with a purity greater than 95% were collected, concentrated and desalted by reverse phase HPLC (0-80% gradient of ACN). The final desalting product was lyophilized from water.

Table E-4 Outline of oligonucleotide synthesis in the DNA / RNA synthesizer ABI-394 used for the synthesis of Example 18-21.

General Purification Method of Examples 18-21:
Buffer A: 20 mM phosphate pH = 6.0 (adjusted with phosphoric acid)
Buffer B: ACN
Column: XBridge Prep C ₁₈ , 5 μm, C ₁₈ , 250 × 10 mm, product number # 186003256
Buffer solution heater set temperature = 50 ° C
Monitor signals at 254 and 280 nm Gradients used:

Analysis of oligonucleotides HPLC method HPLC method 1:
Buffer A: 20 mM phosphate pH = 6.0 (adjusted with phosphoric acid)
Buffer B: ACN
Column: XBridge C ₁₈ , 3.5 μm, C ₁₈ , 4.6 × 50 mm, product number # 186003034
Buffer solution heater set temperature = 35 ° C
Monitor signals at 254 and 280 nm Gradients used:

ＨＰＬＣ方法３：

ＨＰＬＣ方法４：

HPLC method 2:
Buffer A: 50 mM TEAA, pH = 7.8
Buffer B: ACN
Column: XBridge C ₁₈ , 3.5 μm, C ₁₈ , 4.6 × 50 mm, product number # 186003034
Buffer solution heater set temperature = 60 ° C
Monitor signals at 254 and 280 nm Gradients used:

HPLC method 3:

HPLC method 4:

Example 18 Synthesis of Oligonucleotide 122 (All- (Rp) -d [Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1Cs1As1Cs1C] (s1 is as shown below)

Oligonucleotide 122 was synthesized as described above. RT in RP-HPLC (HPLC method 1): 15.2 minutes. UPLC / ESI-MS: C ₃₀₅ H ₄₅₅ N ₈₆ O ₁₂₁ P ₁₉ S ₁₉ calculated value: 8460.25; measured value: 8462.0.

Example 19 Oligonucleotide 123 ((Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp) -d [Gs1Cs1Cs1Ts1Cs1Cs1S1Ts1S1Ts1Ts1Ts1Ts1Ts1Ts1Ts1Ts1Ts1Ts1Ts1Ts1 ] (Synthesis of (1S-17R-1S)

Oligonucleotide 123 was synthesized as described above. RT in RP-HPLC (HPLC method 1): 16.2 minutes. UPLC / ESI-MS: C ₃₀₅ H ₄₅₅ N ₈₆ O ₁₂₁ P ₁₉ S ₁₉ Calculated value: 8460.3; Measured value: 8461.5.

Example 20 Synthesis of Oligonucleotide 124 (All- (Sp) -d [Gs1Cs1Cs1Ts1Cs1As1Gs1Ts1Cs1Ts1Gs1Cs1Ts1Ts1Cs1Gs1Cs1As1Cs1C])

Oligonucleotide 124 was synthesized as described above. RT in RP-HPLC (HPLC method 1): 18.3 minutes. UPLC / ESI-MS: C ₃₀₅ H ₄₅₅ N ₈₆ O ₁₂₁ P ₁₉ S ₁₉ Calculated value: 8460.3; Measured value: 8461.8.

Example 21 Synthesis of oligonucleotide 125 (All- (Rp) -d [5mCs1As1Ts1G])

Oligonucleotide 125 was synthesized as described above. RT in RP-HPLC (HPLC method 2): 16.32 minutes. UPLC / ESI-MS: C ₅₈ H ₈₅ N ₁₈ O ₂₂ P ₃ S ₃ calculated value: 1575.5; measured value: 1575.2.

In Examples 22 and 42, oligonucleotides containing sterically defined methoxyethyl phosphorothioate triester nucleotide linkages were oxalylated to a 1 μmol synthetic column and HCP according to the cycle summarized in Table E-5. It was synthesized with an ABI-394 DNA / RNA synthesizer using 0.8 μmol of dC. The synthesis cycle was carried out with the terminal 5'-O-DMTr group removed (DMT Off). The solid support was washed with dehydrated ACN and dried under a stream of argon. The dried HCP was placed in a plastic vial and treated with 1 mL of dehydrated propylamine (ratio 1: 4) in dehydrated pyridine for 18 hours at room temperature. The solvent was then evaporated and the residue was resuspended in a 10% DMSO-containing aqueous solution with a pH of about 1.5 and the HCP carrier was filtered off. The crude product is purified by reverse phase preparative HPLC (5-65% gradient of ACN in 20 mM sodium phosphate buffer, pH = 6.0). Fractions with a purity greater than 95% are collected, concentrated and desalted by reverse phase HPLC (0-80% gradient of ACN). The final desalting product is lyophilized from water.

Table E-5 Overview of oligonucleotide synthesis in the DNA / RNA synthesizer ABI-394 used for the synthesis of Examples 22 and 42

Example 22 Synthesis of oligonucleotide 126 (All- (Rp) -d [Cs2As2Gs2T] (s2 is as shown below)

Oligonucleotide 126 was synthesized as described above. RT in RP-HPLC (HPLC method 2): 16.23 minutes. UPLC / ESI-MS: C ₄₈ H ₆₈ N ₁₅ O ₂₂ P ₃ S ₃ calculated value: 1396.2; measured value: 1395.2.

Example 23: Oligonucleotides containing sterically defined bulky N-methylpiperadinoester phosphorothioate triester nucleotide-to-nucleotide linkages according to the cycle summarized in Table E-6 on a 1 μmol synthetic column and HCP. The oxalyl-bound 1.5 μmol dT was used for synthesis on the ABI-394 DNA / RNA synthesizer. The synthesis cycle was carried out with the terminal 5'-O-DMTr group removed (DMT Off). The solid support was washed with dehydrated ACN and dried under a stream of argon. The dried HCP was placed in a plastic vial and treated with 1 mL of dehydrated propylamine (ratio 1: 4) in dehydrated pyridine for 18 hours at room temperature. The solvent was then evaporated and the residue was resuspended in a 10% DMSO-containing aqueous solution with a pH of about 1.5 and the HCP carrier was filtered off. The crude product was purified by reverse phase preparative HPLC (5-65% gradient of ACN in 20 mM sodium phosphate buffer, pH = 6.0). Fractions with a purity greater than 95% were collected, concentrated and desalted by reverse phase HPLC (0-80% gradient of ACN). The final desalting product was lyophilized from water.

Table E-6 Outline of oligonucleotide synthesis in the DNA / RNA synthesizer ABI-394 used for the synthesis of Example 23.

Example 23 Synthesis of oligonucleotide 127 (All- (Rp) -d [Cs3As3Gs3T] (s3 is as shown below)

Oligonucleotide 127 was synthesized as described above. RT in RP-HPLC (HPLC method 2): 20.24 minutes. UPLC / ESI-MS: C ₇₈ H ₁₂₂ N ₂₁ O ₂₅ P ₃ S ₃ calculated value: 1943.1; measured value: 1941.0.

Examples 24 and 43: Oligonucleotides containing sterically defined bulky morpholinoester phosphorothioate triester nucleotide-to-nucleotide linkages are bound to HCP with oxalyl on a 1 μmol synthetic column according to the cycle summarized in Table E-7. It was synthesized with an ABI-394 DNA / RNA synthesizer using 1.5 μmol of dT. The synthesis cycle was carried out with the terminal 5'-O-DMTr group removed (DMT Off). The solid support was washed with dehydrated ACN and dried under a stream of argon. The dried HCP was placed in a plastic vial and treated with 1 mL of dehydrated propylamine (ratio 1: 4) in dehydrated pyridine for 18 hours at room temperature. The solvent was then evaporated and the residue was resuspended in a 10% DMSO-containing aqueous solution with a pH of about 1.5 and the HCP carrier was filtered off. The crude product is purified by reverse phase preparative HPLC (5-65% gradient of ACN in 20 mM sodium phosphate buffer, pH = 6.0). Fractions with a purity greater than 95% are collected, concentrated and desalted by reverse phase HPLC (0-80% gradient of ACN). The final desalting product is lyophilized from water.

Table E-7 Overview of oligonucleotide synthesis in the DNA / RNA synthesizer ABI-394 used for the synthesis of Examples 24 and 43

Example 24 Synthesis of Oligonucleotide 128 (All- (Sp) -d [Cs4As4Gs4T] (s4 is as shown below)

Oligonucleotide 128 was synthesized as described above. RT in RP-HPLC (HPLC method 3): 19.75 minutes. UPLC / ESI-MS: C ₇₅ H ₁₁₃ N ₁₈ O ₂₈ P ₃ S ₃ calculated value: 1902.9; measured value: 1904.0.

Example 25: Oligonucleotides containing sterically defined dimethylaminoethyl phosphorothioate triester nucleotide linkages according to the cycles summarized in Table E-8 were oxalyl-linked to a 1 μmol synthetic column and HCP 1.5 μmol. Was synthesized with an ABI-394 DNA / RNA synthesizer using the dT of. The synthesis cycle was carried out with the terminal 5'-O-DMTr group removed (DMT Off). The solid support was washed with dehydrated ACN and dried under a stream of argon. The dried HCP was placed in a plastic vial and treated with 1 mL of dehydrated propylamine (ratio 1: 4) in dehydrated pyridine for 18 hours at room temperature. The solvent was then evaporated and the residue was resuspended in a 10% DMSO-containing aqueous solution with a pH of about 1.5 and the HCP carrier was filtered off. The crude product was purified by reverse phase preparative HPLC (5-65% gradient of ACN in 20 mM sodium phosphate buffer, pH = 6.0). Fractions with a purity greater than 95% were collected, concentrated and desalted by reverse phase HPLC (0-80% gradient of ACN). The final desalting product was lyophilized from water.

Table E-8 Overview of oligonucleotide synthesis in the DNA / RNA synthesizer ABI-394 used for the synthesis of Example 25

Example 25 Synthesis of oligonucleotide 129 (All- (Sp) -d [Cs5As5Gs5T] (s5 is as shown below)

Oligonucleotide 129 was synthesized as described above. RT in RP-HPLC (HPLC method 2): 17.25 minutes. UPLC / ESI-MS: C ₅₁ H ₇₇ N ₁₈ O ₁₉ P ₃ S ₃ calculated value: 1435.4; measured value: 1435.0.

Example 26: According to the cycle summarized in Table E-9, an oligonucleotide containing a sterically defined dimethylalanine ester phosphorothioate triester nucleotide-to-nucleotide bond was attached to a 1 μmol synthetic column and 1.5 μmol to HCP with oxalyl. Was synthesized on the ABI-394 DNA / RNA synthesizer using the dT of. The synthesis cycle was carried out with the terminal 5'-O-DMTr group removed (DMT Off). The solid support was washed with dehydrated ACN and dried under a stream of argon. The dried HCP was placed in a plastic vial and treated with 1 mL of dehydrated propylamine (ratio 1: 4) in dehydrated pyridine for 18 hours at room temperature. The solvent was then evaporated and the residue was resuspended in a 10% DMSO-containing aqueous solution with a pH of about 1.5 and the HCP carrier was filtered off. The crude product is purified by reverse phase preparative HPLC (5-65% gradient of ACN in 20 mM sodium phosphate buffer, pH = 6.0). Fractions with a purity greater than 95% are collected, concentrated and desalted by reverse phase HPLC (0-80% gradient of ACN). The final desalting product is lyophilized from water.

Table E-9 Overview of oligonucleotide synthesis in the DNA / RNA synthesizer ABI-394 used for the synthesis of Example 26

Example 26 Synthesis of Oligonucleotide 130 (All- (Sp) -d [Cs6As6Gs6T] (s6 is as shown below)

Oligonucleotide 130 was synthesized as described above. RT in RP-HPLC (HPLC method 2): 16.45 minutes. UPLC / ESI-MS: C ₅₇ H ₈₃ N ₁₈ O ₂₅ P ₃ S ₃ calculated value: 1609.5; measured value: 1609.6.

Examples 27 and 28: Oligonucleotides containing sterically defined S-methylphosphorothioate triester nucleotide linkages were oxalylated to a 1 μmol synthetic column and 0 according to the cycle summarized in Table E-10. It was synthesized with an ABI-394 DNA / RNA synthesizer using .8 μmol of dC. The synthesis cycle was carried out with the terminal 5'-O-DMTr group removed (DMT Off). The solid support was washed with dehydrated ACN and dried under a stream of argon. The dried HCP was placed in a plastic vial and treated with 1 mL of dehydrated propylamine (ratio 1: 4) in dehydrated pyridine for 18 hours at room temperature. The solvent was then evaporated and the residue was resuspended in a 10% DMSO-containing aqueous solution with a pH of about 1.5 and the HCP carrier was filtered off. The crude product is purified by reverse phase preparative HPLC (5-65% gradient of ACN in 20 mM sodium phosphate buffer, pH = 6.0). Fractions with a purity greater than 95% are collected, concentrated and desalted by reverse phase HPLC (0-80% gradient of ACN). The final desalting product is lyophilized from water.

Table E-10 Overview of oligonucleotide synthesis in the DNA / RNA synthesizer ABI-394 used for the synthesis of Examples 27 and 28

Example 27 Synthesis of Oligonucleotide 131 (All- (Rp) -d [Gs7Cs7Cs7Ts7Cs7As7Gs7Ts7Cs7Ts7Gs7Cs7Ts7Ts7Cs7Gs7Cs7As7Cs7C] (s7 is as shown below)

Oligonucleotide 131 was synthesized as described above. RT on RP-HPLC: 27.65 minutes. UPLC / ESI-MS: C ₂₁₀ H ₂₈₄ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6576.71; measured value: 6575.6.

Example 28 Synthesis of Oligonucleotide 132 (All- (Sp) -d [Gs7Cs7Cs7Ts7Cs7As7Gs7Ts7Cs7Ts7Gs7Cs7Ts7Ts7Cs7Gs7Cs7As7Cs7C] (s7 is as shown below)

Oligonucleotide 132 was synthesized as described above. RT on RP-HPLC: 32.65 minutes. UPLC / ESI-MS: C ₂₁₀ H ₂₈₄ N ₆₇ O ₁₀₂ P ₁₉ S ₁₉ calculated value: 6576.71; measured value: 6574.8.

Synthesis of Chirally Controlled Oligonucleotides Containing Modified Nucleobases As generally described above, and generally as described herein, in some embodiments, the invention is an oligo other than A, T, C and G. Provided are chirally controlled oligonucleotides containing nucleotides. In some embodiments, such chirally controlled oligonucleotides include 5-methylcytosine (5 mC). 21 and the following are non-limiting examples.

Examples 29-41 were oxalylated to a 1 μmol synthetic column and 1.75 μmol dG bound to HCP according to the synthetic cycle summarized in Table E-11 using automated synthesis in the ABI-394 DNA / RNA synthesizer. Was synthesized using. The synthesis cycle was carried out with the terminal 5'-O-DMTr group removed (DMT Off). After completion of automated oligonucleotide synthesis, the HCP carrier was washed with dehydrated ACN and dried under vacuum. The dried HCP was placed in a plastic vial and treated with 1 mL of dehydrated propylamine (ratio 1: 4) in dehydrated pyridine for 18 hours at room temperature. The solvent was then evaporated and the residue was resuspended in a 10% DMSO-containing aqueous solution with a pH of about 1.5 and the HCP carrier was filtered off. The crude product is purified by reverse phase preparative HPLC (following the procedure below). Fractions with a purity greater than 95% are collected, concentrated and desalted by reverse phase HPLC (0-30% gradient of ACN). The final desalting product is lyophilized from water.

Table E-11 Outline of oligonucleotide synthesis in the DNA / RNA synthesizer ABI-394 used for the synthesis of Examples 29-41.

General Purification Method of Examples 29-41:
Buffer A: 20 mM phosphate pH = 6.0 (adjusted with phosphoric acid)
Buffer B: ACN
Column: XBridge Prep C ₁₈ , 5 μm, C ₁₈ , 250 × 10 mm, product number # 186003256
Buffer solution heater set temperature = 50 ° C
Monitor signals at 254 and 280 nm Gradients used:

Example 29 Synthesis of Oligonucleotide 135 (All- (Rp) -d [5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G])

Oligonucleotide 135 was synthesized as described above. RT in RP-HPLC (HPLC method 1): 17.50 minutes. UPLC / ESI-MS: C ₁₈₆ H ₂₇₈ N ₅₁ O ₇₃ P ₁₁ S ₁₁ Calculated value: 5090.0; Measured value: 5091.9.

Example 30 Synthesis of Oligonucleotide 136 (All- (Sp) -d [5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G])

Oligonucleotide 136 was synthesized as described above. RT in RP-HPLC (HPLC method 1): 19.25 minutes. UPLC / ESI-MS: C ₁₈₆ H ₂₇₈ N ₅₁ O ₇₃ P ₁₁ S ₁₁ Calculated value: 5090.0; Measured value: 5090.8.

Example 31 Oligonucleotide 137 ((Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp) -d [5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (1S-9R-1S)

Oligonucleotide 137 was synthesized as described above. RT in RP-HPLC (HPLC method 1): 17.85 minutes. UPLC / ESI-MS: C ₁₈₆ H ₂₇₈ N ₅₁ O ₇₃ P ₁₁ S ₁₁ Calculated value: 5090.0; Measured value: 5091.9.

Example 32 Oligonucleotide 138 ((Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp) -d [5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (2S-7R-2S)

Oligonucleotide 138 was synthesized as described above. RT in RP-HPLC (HPLC method 1): 18.10 minutes. UPLC / ESI-MS: C ₁₈₆ H ₂₇₈ N ₅₁ O ₇₃ P ₁₁ S ₁₁ Calculated value: 5090.0; Measured value: 5091.9.

Example 33 Oligonucleotide 139 ((Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp) -d [5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (1R-9S-1R)

Oligonucleotide 139 was synthesized as described above. RT in RP-HPLC (HPLC method 1): 18.75 minutes. UPLC / ESI-MS: C ₁₈₆ H ₂₇₈ N ₅₁ O ₇₃ P ₁₁ S ₁₁ Calculated value: 5090.0; Measured value: 5088.9.

Example 34 Oligonucleotide 140 ((Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp) -d [5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (2R-7S-2R)

Oligonucleotide 140 was synthesized as described above. RT in RP-HPLC (HPLC method 1): 18.72 minutes. UPLC / ESI-MS: C ₁₈₆ H ₂₇₈ N ₅₁ O ₇₃ P ₁₁ S ₁₁ Calculated value: 5090.0; Measured value: 5091.3.

Example 35 Oligonucleotide 141 ((Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp) -d [5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (3S-5R-3S)

Oligonucleotide 141 was synthesized as described above. RT in RP-HPLC (HPLC method 1): 18.09 minutes. UPLC / ESI-MS: C ₁₈₆ H ₂₇₈ N ₅₁ O ₇₃ P ₁₁ S ₁₁ Calculated value: 5090.0; Measured value: 5090.9.

Example 36 Oligonucleotide 142 ((Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp) -d [5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] (3R-5S-3R)

Oligonucleotide 142 was synthesized as described above. RT in RP-HPLC: 18.35 minutes. UPLC / ESI-MS (HPLC method 1): C ₁₈₆ H ₂₇₈ N ₅₁ O ₇₃ P ₁₁ S ₁₁ calculated value: 5090.0; measured value: 5088.9.

Example 37 Oligonucleotide 143 ((Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp) -d [5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] ((SSR) ₃ -SS)

Oligonucleotide 143 was synthesized as described above. RT in RP-HPLC (HPLC method 1): 18.48 minutes. UPLC / ESI-MS: C ₁₈₆ H ₂₇₈ N ₅₁ O ₇₃ P ₁₁ S ₁₁ Calculated value: 5090.0; Measured value: 5092.0.

Example 38 Oligonucleotide 144 ((Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp) -d [5mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1G] ((RRS) ₃ -R)

Oligonucleotide 144 was synthesized as described above. RT in RP-HPLC (HPLC method 1): 18.02 minutes. UPLC / ESI-MS: C ₁₈₆ H ₂₇₈ N ₅₁ O ₇₃ P ₁₁ S ₁₁ Calculated value: 5090.0; Measured value: 5091.4.

Example 39 Synthesis of Oligonucleotide 145 (All- (Rp) -d [5mCs1Ts15mCs1As1Gs1Ts15mCs1Ts1Gs15mCs1Ts1Ts15mCs1Gs15mC])

Oligonucleotide 145 was synthesized as described above. RT in RP-HPLC (HPLC method 1): 17.30 minutes. UPLC / ESI-MS: C ₂₃₄ H ₃₅₂ N ₆₂ O ₉₂ P ₁₄ S ₁₄ Calculated value: 6388.3. Measured value: 6390.6.

Example 40 Synthesis of oligonucleotide 146 (All- (Rp) -d [Gs15mCs1Ts1G])

Oligonucleotide 146 was synthesized as described above. RT in RP-HPLC (HPLC method 2): 15.89 minutes. UPLC / ESI-MS: C ₅₈ H ₈₅ N ₁₈ O ₂₃ P ₃ S ₃ calculated value: 1591.5; measured value: 1590.8.

Example 41 Synthesis of oligonucleotide 147 (All- (Rp) -d [5mCs1As1Gs1T])

Oligonucleotide 147 was synthesized as described above. RT in RP-HPLC (HPLC method 2): 16.30 minutes. UPLC / ESI-MS: C ₅₈ H ₈₅ N ₁₈ O ₂₂ P ₃ S ₃ calculated value: 1575.5; measured value: 1575.2.

Example 42 Synthesis of Oligonucleotide 148 (All- (Rp) -d [5mCs2As2Gs2Ts25mCs2Ts2Gs25mCs2Ts2Ts25mCs2G])

Oligonucleotide 148 was synthesized as described above using the sulfurizing agent of Example 22. RT in RP-HPLC (HPLC method 3): 16.51 minutes. UPLC / ESI-MS: C ₁₅₃ H ₂₂₃ N ₄₀ O ₇₃ P ₁₁ S ₁₁ calculated value: 4484.1; measured value: 4483.0.

Example 43 Synthesis of Oligonucleotide 149 (All- (Rp) -d [5mCs4As4Gs4Ts45mCs4Ts4Gs45mCs4Ts4Ts45mCs4G])

Oligonucleotide 149 was synthesized as described above using the sulfurizing agent of Example 24. RT in RP-HPLC (HPLC method 4): 17.87 minutes. UPLC / ESI-MS: C ₂₅₂ H ₃₈₈ N ₅₁ O ₉₅ P ₁₁ S ₁₁ Calculated value: 6345.6; Measured value: 6346.5.

Synthesis of Chirally Controlled Chiral Oligonucleotides Containing Different Internucleotide Bindings In some embodiments, the invention comprises different nucleotide-to-nucleotide bindings, as generally described above, and generally as described herein. A chiral controlled oligonucleotide is provided. The following non-limiting examples show such chirally controlled oligonucleotides and methods of their synthesis.

Examples 44-45 show the synthesis of chirally controlled chimeric oligonucleotides, including different internucleotide linkages. Oligonucleotides containing internucleotide bonds mixed with pure morpholinoethyl phosphorothioate triesters / phosphorothioate diastereomers as diastereomers on an ABI-394 DNA / RNA synthesizer according to the cycle summarized in Table E-12, 1 μmol synthetic column. Was synthesized using 1.5 μmol of dT bound to HCP with oxalyl. Two different thiosulfonates were used because different sulfurizing agents can be reacted in each iteration cycle. The synthesis was carried out by removing the terminal 5'-O-DMTr group (DMT Off). The solid support was washed with dehydrated ACN and dried under a stream of argon. The dried solid carrier was then combined with 5 mL of an anhydrous 1M solution of 1,5-diazabicyclo (4.3.0) nona-5-ene (DBN) in dehydrated ACN-trimethylsilyl chloride-16: 1 (v / v). , Treated at room temperature for 10 minutes. The DBN solution was slowly extruded through the column with a plastic luer syringe fixed at the column outlet. The carrier was then washed with dehydrated ACN and dried under vacuum. The dried HCP was placed in a plastic vial and treated with 1 mL of dehydrated propylamine (ratio 1: 4) in dehydrated pyridine for 18 hours at room temperature. The solvent was then evaporated and the residue was resuspended in a 10% DMSO-containing aqueous solution with a pH of about 1.5 and the HCP carrier was filtered off. The crude product is purified by reverse phase preparative HPLC (5-65% gradient of ACN in 20 mM sodium phosphate buffer, pH = 6.0). Fractions with a purity greater than 95% are collected, concentrated and desalted by reverse phase HPLC (0-80% gradient of ACN). The final desalting product is lyophilized from water.

Table E-12 Outline of oligonucleotide synthesis in the DNA / RNA synthesizer ABI-394 used for the synthesis of Examples 44-45.

Example 44 Synthesis of Oligonucleotide 150 (All- (Rp) -d [TsCs1AsT])

Oligonucleotide 150 was synthesized as described above. RT in RP-HPLC (HPLC method 2): 12.72 minutes. UPLC / ESI-MS: C ₄₅ H ₆₂ N ₁₃ O ₂₁ P ₃ S ₃ calculated value: 1310.2; measured value: 1309.2.

Example 45 Synthesis of oligonucleotide 151 (All- (Sp) -d [Cs1AsGs1T])

Oligonucleotide 151 was synthesized as described above. RT in RP-HPLC (HPLC method 2): 14.71 minutes. UPLC / ESI-MS: C ₅₁ H ₇₂ N ₁₇ O ₂₁ P ₃ S ₃ calculated value: 1448.4; measured value: 1446.9.

Example 46 describes the synthesis of chiral controlled chimeric oligonucleotides, including both phosphate diester nucleotide-to-nucleotide bonds and modified nucleotide-to-nucleotide bonds. Exemplary oligonucleotides containing internucleotide bonds mixed with morpholinoethyl phosphorothioate triesters / phosphate diesters were combined with a 1 μmol synthetic column on an ABI-394 DNA / RNA synthesizer according to the cycle summarized in Table E-13. It was synthesized using 1.5 μmol of dT bound to HCP with oxalyl. One thiosulfonate reagent was used for one cycle of sulfurization and iodine-accelerated oxidation was used for the other cycle, as different sulfurizing agents or oxidizing agents could be reacted in each repeating cycle. The synthesis was carried out by removing the terminal 5'-O-DMTr group (DMT Off). The solid support was washed with dehydrated ACN and dried under a stream of argon. The dried HCP was placed in a plastic vial and treated with 1 mL of dehydrated propylamine (ratio 1: 4) in dehydrated pyridine for 18 hours at room temperature. The solvent was then evaporated and the residue was resuspended in a 10% DMSO-containing aqueous solution with a pH of about 1.5 and the HCP carrier was filtered off. The crude product was purified by reverse phase preparative HPLC (5-65% gradient of ACN in 20 mM sodium phosphate buffer, pH = 6.0). Fractions with a purity greater than 95% were collected, concentrated and desalted by reverse phase HPLC (0-80% gradient of ACN). The final desalting product was lyophilized from water.

Table E-13 Outline of oligonucleotide synthesis in the DNA / RNA synthesizer ABI-394 used for the synthesis of Example 46.

Example 46 Synthesis of oligonucleotide 152 (All- (Sp) -d [Cs1AGs1T])

Oligonucleotide 152 was synthesized as described above. RT in RP-HPLC (HPLC method 2): 13.42 minutes. UPLC / ESI-MS: C ₅₁ H ₇₂ N ₁₇ O ₂₂ P ₃ S ₂ calculated value: 1432.3; actually measured value: 1431.7.

Example 47 describes the synthesis of chiral-controlled chimeric oligonucleotides, including both phosphate diester nucleotide linkages, as well as both purely modified phosphate triester and phosphate diester nucleotide linkages as chiral. A chiral-controlled oligonucleotide containing a mixed bond of mixed morpholinoethyl phosphorothioate triesters / phosphate diesters / phosphodiester diesters was administered on an ABI-394 DNA / RNA synthesizer in an ABI-394 DNA / RNA synthesizer according to the cycle summarized in Table E-14, 1 μmol. It was synthesized using a synthetic column and 1.5 μmol of dT bound to HCP with oxalyl. Since different sulfurizing agents or oxidizing agents can be reacted in each repeating cycle, two different thisulfonate reagents were used in two different sulfurizing cycles and iodine-accelerated oxidation was used in the other cycle. The synthesis was carried out by removing the terminal 5'-O-DMTr group (DMT Off). The solid support was washed with dehydrated ACN and dried under a stream of argon. The dried solid carrier was then combined with 5 mL of an anhydrous 1M solution of 1,5-diazabicyclo (4.3.0) nona-5-ene (DBN) in dehydrated ACN-trimethylsilyl chloride-16: 1 (v / v). , Treated at room temperature for 10 minutes. The DBN solution was slowly extruded through the column with a plastic luer syringe fixed at the column outlet. The carrier was then washed with dehydrated ACN and dried under vacuum. The dried HCP was placed in a plastic vial and treated with 1 mL of dehydrated propylamine (ratio 1: 4) in dehydrated pyridine for 18 hours at room temperature. The solvent was then evaporated and the residue was resuspended in a 10% DMSO-containing aqueous solution with a pH of about 1.5 and the HCP carrier was filtered off. The crude product was purified by reverse phase preparative HPLC (5-65% gradient of ACN in 20 mM sodium phosphate buffer, pH = 6.0). Fractions with a purity greater than 95% were collected, concentrated and desalted by reverse phase HPLC (0-80% gradient of ACN). The final desalting product was lyophilized from water.

Table E-14 Overview of oligonucleotide synthesis in the DNA / RNA synthesizer ABI-394 used for the synthesis of Example 47

Example 47 Synthesis of Oligonucleotide 153 (All- (Sp) -d [CAs1GsT]) Oligonucleotide 153 was synthesized as described above. RT in RP-HPLC (HPLC method 2): 11.48 minutes. UPLC / ESI-MS: C ₄₅ H ₆₁ N ₁₆ O ₂₁ P ₃ S ₂ calculated value: 1318.1; measured value: 1318.1.

As will be appreciated by those of skill in the art, longer chimeric chiralally controlled oligonucleotides can be prepared according to Examples 46 and 47, as well as other examples and methods described herein.

Chirally pure oligonucleotides have different properties than mixtures of diastereomers by non-stereospecific synthesis, as described above and as described herein, although in some embodiments they have the same base sequence. The present application provides pure as chiral oligonucleotides having different chemical and biological properties from the mixture of diastereomers synthesized by non-stereospecific oligonucleotide synthesis.

Example 48 HPLC properties of a mixture of pure oligonucleotides as chiral and diastereomers by non-stereospecific synthesis

Chirally pure phosphorothioate diester oligonucleotides A (total _RP , oligonucleotide 101) and B (total _SP , oligonucleotide 102), as well as non-stereospecific C made by standard non-stereospecific oligonucleotide synthesis. (Three-dimensionally random phosphorothioate diester nucleotide linkages) were analyzed under the same RP-HPLC conditions and the corresponding HPLC traces are shown in FIG.

As clearly shown in FIG. 2, the stereochemistry of phosphodiester 20-mer oligonucleotides affects their behavior as determined by RP-HPLC and IEX-HPLC. Although not intended to be constrained by theory, a correlation is observed with the retention time (RT) of RP-HPLC and IEX-HPLC because the retention time (RT) trends are preserved. The total _RP stereoisomer (A) has a shorter retention than the total _SP stereoisomer (B) and is a sterically random phosphorothioate diasteronucleotide (a mixture of all ²¹⁹ stereoisomers). C) has a wide HPLC peak and elutes between the extreme total _RP and all _SP diastereomers.

As will be appreciated by those skilled in the art, an analysis showing a comparison of different chiral-controlled or non-chiral-controlled (eg, sterically random) oligonucleotide compositions having the same sequence is an exemplary chiral-uncontrolled oligonucleotide. Both have been shown to show that the composition (ie, prepared by non-chiral controlled oligonucleotide synthesis) contains only very low levels of certain oligonucleotide types, such as total _RP or _SP type. The data confirms.

Example 49 Thermal denaturation experiment (Tm)

Each DNA strand was mixed with complementary RNA at an equimolar concentration of 0.5 μM in 1 × PBS. A total 2.5 mL solution was prepared for each double strand and the mixture was heated at 90 ° C. for 2 minutes and allowed to cool for several hours. The mixture was then stored at 4 ° C. for 2 hours. Using a PerkinElmer UV spectrophotometer equipped with a Pelche unit, the temperature gradient starts from 15 ° C. and rises to 90 ° C. at 0.5 ° C./min intervals at 0.5 minute intervals. Absorbance at 254 nm was recorded. The absorbance at 254 nm was plotted against temperature and the Tm value was calculated from the respective linear derivative of each curve.

FIG. 3 shows the difference in Tm between two sterically pure diastereoisomer phosphorothioate oligonucleotides (total _RP 20-mer A and total _SP 20-mer B) and sterically random phosphorothioate oligonucleotide C. Is shown. Total _RP phosphorothioate DNA shows higher affinity for complementary RNA when compared to both the opposite total SP diastereoisomer and the sterically random 20 _- mer.

Table E-15 below summarizes the differences between chiral-controlled oligonucleotides and sterically random oligonucleotides.

Table E-15 Differences between chirally controlled oligonucleotides and sterically random oligonucleotides

Example 50 Biological activity of pure oligonucleotides as chiral

After confirming OD, all oligonucleotide candidate molecules were diluted to a starting concentration of 20 μM. Multidose forward transfection experiments, 96 wells using Hep3B cells (ATCC®, Catalog HB-8604®) and Lipofectamine 2000 (Life Technologies, Catalog 11668-019) transfection reagents. Set on the plate.

Transfection protocol:

2 × 10 ⁴ Hep3Bs per well are seeded and contains 100 μl of antibacterial agent containing 10% FBS and 1% Glutamax I (Life Technologies, Catalog 35050061) in a CO ₂ incubator at 37 ° C. for 24 hours. Incubated in no MEM medium (Life Technologies, Catalog 11095098). Twelve dilutions were set in nuclease-free water (Table E-16).

Table E-16 Concentrations of oligonucleotide transfection stock plates

In each well, 0.5 μL Lipofectamine 2000 was mixed with 9.5 μL Opti MEM I reduced serum medium (Life Technologies, Catalog 31985062) and incubated for 5 minutes. After incubation, each candidate at the reported concentration of 10 μL was mixed with diluted Lipofectamine 2000 by gentle pipetting. The mixture was then incubated at room temperature for 20 minutes to form a complex. During this time, the cell growth medium was replaced with 80 μL of the new antibacterial-free MEM. 20 μL of lipid-oligo complex was gently mixed into each well to give a total volume of 100 μL. The cells were then incubated at 37 ° C. for 24 hours in a CO ₂ incubator.

RNA extraction:

After 24 hours of incubation, the cell growth medium was removed and RNA was extracted using the Dynabeads mRNA Direct Kit (Life Technologies, Catalog 61012) according to the procedure provided in the kit manual without modification. This magnetic Oligo (dT) ²⁵ bead system enables cost-effective, durable high-throughput poly (A) RNA extraction and avoids DNAse treatment. RNA was eluted with nuclease-free water and stored at −80 ° C.

cDNA synthesis:

A high volume cDNA reverse transcription kit (Life Technologies, Catalog 43749667) was used and 10 μL RNA was used in a 20 μL cDNA synthesis reaction using an RNAse inhibitor and the kit protocol. Reverse transcription was performed on a C1000 Touch thermal cycler (Biorad, Catalog 185-1196EDU) in 96-well format.

Gene expression analysis:

Gene expression was measured by quantitative PCR on a LightCycler 480 real-time PCR device using a LightCyclor® 480 SYBR Green I Master (Roche, Catalog 04707516001). A primer for the human sequence of target apolipoprotein B (Apo B) (NM_000384) and an endogenous control glyceraldehyde 3-phosphate dehydrogenase (GM_002046) were designed and ordered from IDT (Table E-17).

Table E-17 Sequence of HPLC purified primers used for quantifying gene expression

Measurements of targets and endogenous controls were performed in separate wells using substances from the same cDNA template. The SYBR green assay PCR conditions were set as described in the SYBR Green I Master protocol (Table E-18).
Table E-18 Real-time PCR conditions for SYBR green assay

Melting curve analysis shows a single unit replication sequence peak for each primer pair (Fig. 4).

The data are the result of three biological replications. All samples were compared to untreated untransfected controls. The following results were observed when gene expression was evaluated by real-time PCR using SYBR green (Table E-19, FIG. 5).

Complete _IC50 data evaluated by Table E-19 SYBR Green

In some embodiments, the chiral controlled oligonucleotide provided exhibits an _IC50 of less than 8 nM. In some embodiments, the chiral controlled oligonucleotide provided exhibits an _IC50 of less than 7 nM. In some embodiments, the chiral controlled oligonucleotide provided exhibits an _IC50 of less than 6 nM. In some embodiments, the chiral controlled oligonucleotides provided exhibit an _IC50 of less than 5 nM. In some embodiments, the chiral controlled oligonucleotides provided exhibit an _IC50 of less than 4 nM. In some embodiments, the chiral controlled oligonucleotides provided exhibit an _IC50 of less than 3 nM. In some embodiments, the chiral controlled oligonucleotides provided exhibit an _IC50 of less than 2 nM. In some embodiments, the chiral controlled oligonucleotides provided exhibit an _IC50 in the range of 0.5-8 nM. In some embodiments, the chiral controlled oligonucleotides provided exhibit IC _50s in the range of 1-4 nM. In some embodiments, the chiral controlled oligonucleotides provided exhibit an _IC50 in the range of 1.5-3 nM. In some embodiments, the chiral controlled oligonucleotides provided exhibit an _IC50 in the range of 1.5-2 nM. In some embodiments, the chiral controlled oligonucleotides provided exhibit an _IC50 in the range of 0.5-2 nM. In some embodiments, the chiral controlled oligonucleotides provided exhibit IC _50s in the range of 1-2.5 nM. In some embodiments, the chiral controlled oligonucleotides provided exhibit an _IC50 in the range of 1.5-3 nM. In some embodiments, the chiral controlled oligonucleotides provided exhibit an _IC50 in the range of 2.5-5 nM. In some embodiments, the chiral controlled oligonucleotides provided exhibit IC _50s in the range of 3-6 nM. In some embodiments, the chiral controlled oligonucleotides provided exhibit IC _50s in the range of 5-8 nM. In some embodiments, the chiral controlled oligonucleotide provided exhibits an _IC50 within the range between the upper and lower boundaries. In some such embodiments, the upper boundary is 8, 5, 4, or 3 nM. In some such embodiments, the lower boundary is 1, 1.5, 2, or 2.5 nM. As can be seen by reference to the examples, the present disclosure illustrates in detail a variety of chiral-controlled oligonucleotides, or chiral-controlled oligonucleotide compositions, exhibiting such representative _IC50 values.

Table E-19 shows that when tested in a chiral controlled form, a particular chiral controlled oligonucleotide / oligonucleotide composition is more effective than the corresponding sterically random oligonucleotide mixture (with oligonucleotide 118). By comparison, oligonucleotides 105, 109, 115 and 116); they have also been shown to be more active than the sterically random oligonucleotide mixture mipomersen (oligonucleotide 104 compared to oligonucleotide 120). , 105, 106, 107, 109, 115 and 116).

Further Chiral Controlled Formula Example 51 Preparation of Chiral Controlled Oligonucleotide Formulation

This example describes the preparation of a variety of specific chiral controlled compositions of the particular oligonucleotides described herein. In particular, this example describes oligonucleotide preparation using loaded lcaa-CPG-500.

N ⁴ -Bz-5'-O-DMTr-2'-O-MOE-5mC (1) (4.0 g, 5.5 mmol) was dissolved in anhydrous DCM (20 mL) and 2 equivalents of succinic anhydride (1). .1 g, 11.1 mmol), and 3 equivalents of 4-N, N-dimethylaminopyridine (2.0 g, 16.6 mmol). The reaction was stirred under argon at room temperature. After the starting material has been completely consumed (1 hour) as determined by TLC, the solvent is evaporated to dry and the crude residue is dissolved in DCM containing 1% triethylamine and then 2%. Purification by flash silica gel chromatography using a 0-2% gradient of MeOH in DCM containing triethylamine. The yield of the high-purity compound (2) after evaporation was 4.5 g, 88%. The resulting 3'-O-succinate (2) (0.92 g, 1.0 mmol), N, N-diiropropylethylamine (0.82 ml, 5.0 mmol) and CPG-500 (10 g) were added to DMF (10 g). It was placed in 50 mL), and then HBTU (0.42 g, 1.1 mmol) was added. The mixture was shaken for 2 hours and then filtered. The carriers were washed with DMF, MeOH and finally DCM and then dried under vacuum. Trityl cation analysis (monitored at 504 nm) showed that the loading of nucleosides on carrier (3) was 63 μmol / g.

A chiral-controlled oligonucleotide composition containing a sterically defined chimeric deoxy and a 2'-O-MOE phosphorothioate triester nucleotide bond was prepared in Table E-20 on a MerMade-12 DNA / RNA synthesizer (BioAutomation). Uncapped N4-Bz-5'-O-DMTr-2'-O-MOE- ^5mC (63 μmol / g, Glen Research lcaa CPG) bound with 2.0 g (126 μmol) succinyl according to the cycle summarized in. -500) was packed using an MM-6-200 synthetic column (BioAutomation) for synthesis. The oligonucleotide synthesis cycle was performed in the preliminary capping step (capping 2) without removal of the final terminal 5'-O-DMTr oligonucleotide group (DMT On). The stereospecific sulfurization step was performed using a 0.3 M methylthiosulfonic acid S- (2-cyanoethyl) reagent after the corresponding chiral phosphoramide coupling and the two-step capping step (Table E-20).

When the automated oligonucleotide synthesis cycle was completed with the final 5'-O-DMTr group attached, the synthetic column was removed from the DNA / RNA synthesizer and dried under vacuum. The dried carrier was transferred to an empty glass manual peptide synthesizer and in a 40 mL solution 0.5 M 1,5-diazabicyclo (4.3.0) nona-5-ene (DBN), 0.25 M N, O in ACN. -Bis (trimethylsilyl) trifluoroacetamide (BSTFA) was continuously passed through the carrier in a manual peptide synthesizer for 5 minutes without stopping the flow. The carrier was washed with 50% Py / ACN and dried under a stream of argon for 1 second. The carrier was then transferred to an empty screw-capped plastic vial, treated with 5% EtOH / concentrated NH ₃ (20 mL) at 60 ° C. for 6 hours and left at room temperature for 12 hours. The carrier was removed by filtration and washed with concentrated NH ₃ . The filtrate was dried under vacuum and then the product was dissolved in 50 mM TEAA (120 mL). If there were suspended matter in the solution, further filtration was performed. The solution was filled in a Sep-Pak cartridge (Waters, Sep-Pak Vac 35cc (10g) C ₁₈ cartridge). The cartridge was washed with 20% ACN / 50 mM TEAA (70 mL), the capped total truncated sequence was removed, and the full length DMT On oligonucleotide was removed with 50% ACN / water (50 mL) containing 0.5% concentrated NH ₃ . Was eluted. The solution containing the DMT On oligonucleotide is dried under vacuum and then diluted with 50 mM TEAA (120 mL) and another Sep-Pak cartridge (Waters, Sep-Pak Vac 35 cc (10 g) C ₁₈ cartridge). Filled with. The cartridge was washed with Milli-Q water (50 mL), 2% TFA / water (50 mL), then water (50 mL). The DMT Off oligonucleotide was eluted with 50% ACN / water (50 mL) containing 0.5% concentrated NH ₃ .
Table E-20 Overview of oligonucleotide synthesis in the DNA / RNA synthesizer MerMade-12 used for chiral controlled synthesis

Synthesis of oligonucleotide ONT-75 (All (Rp))- Gs5mCs5mCsTs5mCs AsGsTs5mCsTsGs5mCsTsTs5mCs Gs5mCsAs5mCs5mC

Oligonucleotide ONT-80 (All (Sp))-Synthesis of Gs5mCs5mCsTs5mCs AsGsTs5mCsTsGs5mCsTsTs5mCs Gs5mCsAs5mCs5mC

Oligonucleotide ONT-77 (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp , Rp , Rp) -10S-4R) synthesis

Oligonucleotide ONT-81 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp , Sp , Sp) -10R-4S) synthesis

Oligonucleotide ONT-87 (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Sp, Sp, Rp, Rp, Rp, Rp , Rp , Rp) - ₍ SSR) 3-5R) synthesis

Oligonucleotide ONT-88 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp ) -Gs5mCs5mCsTs5mCsAsGsTs5mCs5sCs5sCs5sCs5mCs -(RRS) _3-5S )

Oligonucleotide ONT-89 (Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp , Sp, Rp , Sp) SR) ₉ S) synthesis

Synthesis of oligonucleotide ONT-82 (All (Rp))- GsTs5mCs5mCs5mCs TsGsAsAsGsAsTsGsTs5mCsAsAsTsGs5mC

Synthesis of oligonucleotide ONT-84 (All (Sp))- GsTs5mCs5mCs5mCs TsGsAsAsGsAsTsGsTs5mCsAsAsTsGs5mC

Oligonucleotide ONT-85 (Rp, Rp, Rp, Rp, Rp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Sp, Rp, Rp , Rp , Rp) -10S-4R) synthesis

Oligonucleotide ONT-86 (Sp, Sp, Sp, Sp, Sp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Rp, Sp, Sp , Sp , Sp) -10R-4S) synthesis

Example 52 Common RP-HPLC methods for analysis of crude DMT On oligonucleotides and purified DMT Off oligonucleotides This example is crude and purified by the chiral-controlled synthesis described herein. RP-HPLC analysis of oligonucleotide compositions is described.
Buffer A: 50 mM TEAA, pH = 7.0
Buffer B: ACN
Column: XBridge C ₁₈ , 3.5 μm, C ₁₈ , 4.6 × 50 mm, product number # 186003034
Column temperature = 50 ° C
Monitor signals at 254 and 280 nm Gradients used:

Example 53 General IEX-HPLC Method for Analysis of Crude DMT On Oligonucleotides and Purified DMT Off Oligonucleotides This Example is crude and purified prepared by the chiral-controlled synthesis described herein. IEX-HPLC analysis of oligonucleotide compositions is described.
Buffer A: 10 mM Tris HCl, 50% ACN, pH = 8.0
Buffer B: 10 mM Tris HCl, 800 mM NaClO ₄ , pH = 8.0
Column: DIONEX, DNAPac, PA-100, for analysis, 4.0 x 250 mm, product number # 063000
Column temperature = 60 ° C
Monitor signals at 254 and 280 nm Gradients used:

Example 54 General UPLC-LCMS Method for Analysis of Purified DMT Off Oligonucleotides This example is the UPLC-LCMS of a purified oligonucleotide composition prepared by the chiral-controlled synthesis described herein. Describe the analysis.
Buffer A: 15 mM TEA, 400 mM HFIP, water Buffer B: 50:50 Buffer A / Methanol Column: UPLC @ OST C ₁₈ 1.7 μm, 2.1 × 500 mm
Column temperature = 50 ° C
Gradient used:

Example 55 General IEX-HPLC method for purification of crude DMT Off oligonucleotides

This example describes the IEX-HPLC purification of a crude oligonucleotide composition prepared by the chiral controlled synthesis described herein.
Buffer A: 20 mM NaOH, pH = 11.0
Buffer B: 20 mM NaOH, 2.5 M NaCl, pH = 11.0
Column: Empty column Waters AP-2 (Waters), Source15Q carrier (GE Healthcare) was custom filled in-house. Individual purified columns were packed and used for different sterically pure oligonucleotides.
Equipment: AKTA refiner with P-900 pump, UPC-900 detector, and 50 mL injection SuperLoop (GE Healthcare) Buffer heater temperature setting = 70 ° C.
Monitor signal at 254 nm Fraction capacity: 5 mL
Gradient used:

The recovered fractions were individually analyzed by analytical IEX-HPLC using the above analytical conditions and gradients. High-purity fractions were collected to provide purified material with a purity of 95% or higher as determined by the UV absorption profile at 254 nm.

Table E-21 Summary of physicochemical properties of sterically pure phosphorothioate oligonucleotides synthesized as described in Example XX

Sequence A: Human ApoB sequence 5' -Gs5mCs5mCsTs5mCs AsGsTs5mCsTsGs5mCsTsTs5mCs Gs5mCsAs5mCs5mC -3'.

Sequence B: Mouse ApoB sequence 5' -GsTs5mCs5mCs5mCs TsGsAsAsGsAsTsGsTs5mCs AsAsTsGs5mC -3'.

Underlined nucleotides indicate 2'-O-MOE. s = phosphorothioate bond. 5mc = 5-methyl-2'-deoxycytidine. 5mc = 5-methyl-2'-O-MOE-cytidine. The steric structure describes the stereoisomeric properties ( _RP / _SP ) of each phosphorus atom at a given phosphorothioate bond of the oligonucleotide. Retention time (t _R ) and measured molecular weight (MW) values for IEX-HPLC were obtained using the corresponding analytical methods for purified compounds (above).

Example 56 General RP-HPLC method for overlay analysis of purified DMT Off oligonucleotides This example is the RP of the purified oligonucleotide composition prepared by the chiral-controlled synthesis described herein. -Describe an HPLC analysis.
Buffer A: 50 mM TEAA, pH = 7.0
Buffer B: ACN
Column: XBridge C ₁₈ , 3.5 μm, C ₁₈ , 4.6 × 50 mm
Column temperature = 50 ° C
Monitor signals at 254 and 280 nm Gradients used:

Panel A of FIG. 37 shows purified DMT Off oligonucleotides ONT-75, ONT-77, ONT-80, ONT-81, ONT-87, ONT-88, ONT-89, and ONT-41 (diastereomixture). -A superposition of RP-HPLC traces of Gs5mCs5mCsTs5mCs AsGsTs5mCsTsGs5mCsTsTs5mCs Gs5mCsAs5mCs5mC .

Panel B in FIG. 37 shows an enlarged view of panel A, and each curve is labeled as follows.

Panel A of FIG. 38 is a stack of purified DMT Off oligonucleotides ONT-82, ONT-84, ONT-85, ONT-86, and ONT-83 (diastereomix) -GsTs5mCs5mCs5mCsTsGsAsAsGsAsTsGsTs5mCsAsTsGs5m .

Panel B in FIG. 38 shows an enlarged view of panel A, and each curve is labeled as follows.

Example 57: Thermal denaturation experiment (Tm)

This example describes the properties of a chirally controlled oligonucleotide composition using thermal denaturation.

Each DNA strand was mixed with complementary RNA at an equimolar concentration of 1 μM in 1 × PBS. A total 3 mL solution was prepared for each double strand and the mixture was heated at 90 ° C. for 2 minutes and allowed to cool for several hours. The mixture was then stored at 4 ° C. for 2 hours. Using the Cary100 series (Agilent Technologies), increase at 0.5 ° C./min with a temperature gradient starting from 15.0 ° C. to 95.0 ° C. at 0.5 minute intervals. Absorbance at 254 nm was recorded. The absorbance at 254 nm was plotted against temperature and the Tm value was calculated from the respective linear derivative of each curve.

FIG. 39 shows a Tm superposition of chirally controlled oligonucleotides and sterically random oligonucleotides.

FIG. 39 shows four sterically pure diastereoisomer phosphorothioate oligonucleotides (all- _RP 20-mer, all-SP 20-mer, 5R-10S-4R and 5S-10R-4S _gapmers ). )) And the difference in Tm between sterically random phosphorothioate oligonucleotides. Total _RP phosphorothioate DNA exhibits affinity for the highest complementary RNA (Tm = 85.1 ° C.) compared to total Sp20 mer (Tm = 75.1 ° C.) with the lowest affinity. 5R-10S-4R and 5S-10R-4S gapmers, and sterically random phosphorothioate oligonucleotides all showed intermediate values (Tm range = 80.1-81.2 ° C.).

Table E-22 below shows the human ApoB sequence 5'- Gs5mCs5mCsTs5mCs AsGsTs5mCsTsGs5mCsTsTs5mCs Gs5mCsAs5mCs5mC-3 ', stereophysically pure phosphorothioate oligonucleotides, stereo-stereoscopically pure phosphorothioate Shows various stereochemical structures of.

Table E-22

Example 58 Preparation of an exemplary chiral-controlled SiRNA oligonucleotide target PCSK9 Proprotein convertase subtilisin / kexin type 9 (PCSK9) is an enzyme involved in cholesterol metabolism. PCSK9 binds to the receptor for low-density lipoprotein (LDL) and induces its destruction. When the receptor is disrupted, the LDL associated with the receptor is also removed, but the net effect of bound PCSK9 actually increases LDL levels, which is otherwise to the cell surface. It will return to the cycle and remove more cholesterol.

Several companies are developing therapeutic agents that target PCSK9. Of particular relevance to the present disclosure is that Isis Pharmaceuticals, Santaris Pharmaceuticals and Alnylam Pharmaceuticals are developing nucleic acid agents that inhibit PCSK0. The antisense oligonucleotide, Isis Pharmaceuticals products, have been shown to increase the expression of LDLR and reduce circulating total cholesterol levels in mice (Graham et al. "Antisense inhibition of proteinin protectase subsulin / kicksin". hyperlipidemia ". J. Lipid Res. 48 (4): 763-7, April 2007). Initial clinical trials of Alnylam Pharmaceuticals products in ALN-PCS have shown that RNA interference provides an effective mechanism for inhibiting PCSK9 (Frank-Kamenetsky et al. LDL cholesterol in nonhuman preferences ”. Proc. Natl. Acad. Sci. USA 105 (33): 1195-20, August 2008).

注記：小文字は２’ＯＭｅ ＲＮＡ残基を表し；大文字は２’ＯＨ ＲＮＡ残基を表し；太字および斜体の「ｓ」はホスホロチオエート部を示す。 This example describes various siRNA agents targeting PCSK9 that are chiral controlled or sterically random. In particular, this example describes the preparation of each oligonucleotide composition below, as shown in Table E-23 below.

Table E-23

Note: Lowercase letters represent 2'OMe RNA residues; uppercase letters represent 2'OH RNA residues; bold and italicized "s" indicate phosphorothioate moieties.

The Lcaa-CPG-500 used in the preparation of these oligonucleotides was prepared according to the following reaction.

Specifically, 5'-O-DMTr-2'deoxythymidine (1) (4.28 g, 7.86 mmol) was dissolved in anhydrous DCM (50 mL) and 2 equivalents of succinic anhydride (1.57 g, 15) were dissolved. .7 mmol) and 3 equivalents of 4-N, N-dimethylaminopyridine (2.88 g, 23.6 mmol). The reaction was stirred under argon at room temperature. After the starting material has been completely consumed (1 hour) as determined by TLC, the solvent is evaporated to dry and the crude residue is dissolved in DCM containing 1% triethylamine and then 2%. Purification by flash silica gel chromatography using a 0-2% gradient of MeOH in DCM containing triethylamine. The yield of the high-purity compound (2) after evaporation was 5.5 g, 94%. 5'-O-DMTr-2'-deoxythymidine-3'-O-succinic acid ester (2) (0.60 g, 0.81 mmol) produced, N, N-diiropropylethylamine (0.71 mL, 4) .02 mmol) and CPG-500 (10 g) were placed in DMF (50 mL), then HBTU (0.37 g, 0.97 mmol) was added. The mixture was shaken for 2 hours and then filtered. The carriers were washed with DMF, MeOH and finally DCM and then dried under vacuum. Trityl cation analysis (monitored at 504 nm) showed that the loading of nucleosides on carrier (3) was 38 μmol / g.

As shown in Table E-23 above, each nucleotide containing a 2'-OH and 2'-OMe phosphodiester bond and a sterically defined 2'-deoxy and 2'-OMe phosphorothioate diester nucleotide linkage, ABI394. In a DNA / RNA synthesizer, 5'-O-DMTr-2'-deoxythymidine (38 μmol / g) bound with 130 mg (4.9 μmol) of succinyl according to the cycles summarized in Tables E-24 and E-25, respectively. ) Was packed and synthesized using a synthetic column having a capacity of 10 μmol. Prior to the synthesis, a preliminary capping step (capping 2) was performed to remove the terminal 5'-O-DMTr group to complete the synthesis. The oxidation step was performed using a commercially available 5-6M decane solution of tert-butyl hydroperoxide (TBHP), which was then diluted with 4 parts dichloromethane. The stereospecific sulfide step was performed using a 0.3 M methylthiosulfonic acid S-cyanoethyl reagent after the corresponding chiral phosphoramide coupling and two capping steps (Table E-25).

表Ｅ－２５ ＤＮＡ／ＲＮＡ合成装置ＡＢＩ３９４におけるオリゴヌクレオチド合成の概要（立体的に規定されたホスホロチオエート２’‐デオキシおよび２’‐ＯＭｅ ＲＮＡサイクル）

After the automated oligonucleotide synthesis cycle was completed and the final 5'-O-DMTr group was removed, the synthetic column was removed from the DNA / RNA synthesizer and dried under vacuum. 0.5M 1,5-diazabicyclo (4.3.0) nona-5-ene (DBN) in a 10mL solution, 0.25MN in ACN, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) 1 For minutes, the flow was not stopped and was continuously added to the carrier through the synthetic column using a syringe attached to one end of the synthetic column. The carrier was then washed with anhydrous ACN and dried under vacuum. The dried carrier was then transferred to an empty screw-capped plastic vial and treated with 40% MeNH ₂ aqueous solution (0.5 mL) at 60 ° C. for 10 minutes. The vial was then immediately cooled, diluted with DMSO (0.5 mL), the carrier removed by filtration, washed again with DMSO (1 mL) and filtered. The filtrate was cooled and then immediately treated with _3HF Et 3N (0.75 mL) at 60 ° C. for 10 minutes, then immediately frozen and stored at 4 ° C. before purification.

Table E-24 Outline of oligonucleotide synthesis in DNA / RNA synthesizer ABI394 (2'-O-TBDMS and 2'-OMe substituted RNA cycle)

Table E-25 Outline of oligonucleotide synthesis in DNA / RNA synthesizer ABI394 (sterically defined phosphorothioate 2'-deoxy and 2'-OMe RNA cycle)

Example 59 Common IEX-HPLC method for analysis of crude and produced DMT Off RNA oligonucleotides Buffer A: 20 mM sodium phosphate, pH = 11.0
Buffer B: 20 mM sodium phosphate, 1 M NaBr, pH = 11.0
Column: DIONEX, DNAPac, PA-100, for analysis, 4.0 x 250 mm
Column temperature: 60 ° C
Monitor signals at 254 nm and 280 nm Gradients used:

Example 60 General UPLC-LCMS Method for Analysis of Purified DMT Off RNA oligonucleotides Buffer A: 15 mM TEA, 400 mM HFIP, Water Buffer B: 50:50 Buffer A / Methanol Column: UPLC @ OST C ₁₈ 1.7 μm, 2.1 × 500 mm
Column temperature = 50 ° C
Gradient used:

Example 61 General IEX-HPLC method for purification of crude DMTr Off RNA oligonucleotide Buffer A: 20 mM NaOH, pH = 8.5
Buffer B: 20 mM NaOH, 1M NaOH, pH = 8.5
Column: Empty column Waters AP-2 (Waters), Source15Q carrier (GE Healthcare) was custom self-filled. The same purified column was used for different sterically pure oligonucleotides.
Instrument: Waters HPLC unit with 2525 two-component gradient module, 2487 two-wavelength absorbance detector, and 20 mL Flex injector (Waters).
Buffer heater temperature setting = 70 ° C
Monitor signals at 254 nm and 280 nm Fraction capacity: 4.5 mL
Gradient used:

The recovered fractions were individually analyzed by analytical IEX-HPLC using the above analytical conditions and gradients. High-purity fractions were collected to provide purified material with a purity of 95% or higher as determined by the UV absorption profile at 254 nm.

Example 62 General RP-Sep-Pak method for desalting purified RNA oligonucleotides

The stored pure fractional solution was loaded into a Sep-Pak cartridge (Waters, Sep-Pak Vac 35cc (10g) C ₁₈ cartridge) pre-prepared with water. After filling the sample (100 mL), the cartridge was washed with Milli-Q water (50 mL) to remove all salts and then washed with 50% ACN / water to elute full-length desalted RNA oligonucleotides. The recovered solution was concentrated under vacuum to a volume of 5 mL and lyophilized from water.

Example 63 Preparation of a double-stranded siRNA drug using a chiral-controlled oligonucleotide chain.

This example describes the preparation of a double-stranded siRNA drug by thermal annealing of the chiral-controlled oligonucleotide strands described above.

Each RNA strand was mixed with the complementary RNA strand at an equimolar concentration of 10 μM in 1 × PBS. A total 0.5 mL solution was prepared for each double strand and the mixture was heated at 90 ° C. for 2 minutes and allowed to cool for several hours. The mixture was then stored at 4 ° C. The physicochemical properties of the RNA strand used are shown in Table E-26.
Table E-26

Oligonucleotide sequence used, human PCSK9 siRNA: sense strand (S) 5'-uucuAGACcuGuuuuGcuudTsdT-3'; antisense strand 1 (AS1) 5'-AAGcAAAAacAGGUCuAGAAdTsdT-3'; antisense strand 2 (ASAG-ASAG2 3'. Uppercase nucleotides: RNA, lowercase nucleotides 2'-OMe, d = 2'-deoxy, s = phosphorothioate. The three-dimensional structure describes the stereoisomeric properties ( _RP / _SP absolute configuration) of each phosphorus atom in a given phosphorothioate bond of an oligonucleotide. Measured molecular weight (MW) values were obtained using the corresponding analytical methods for purified compounds (above).

FIG. 51 shows the superposition of IEX-HPLC profiles and shows the difference in retention time between sterically pure RNA oligonucleotides.

FIG. 52 shows the superposition of IEX-HPLC profiles and shows the difference in retention time between sterically pure RNA oligonucleotides.

In addition to the prepared chiral-controlled siRNA oligonucleotides exemplified above in detail, the invention also provides the preparation of siRNA chains having several chiral phosphorothioate nucleotide linkages and total chiral phosphorothioate nucleotide linkages.

注記：小文字は２’‐ＯＭｅ ＲＮＡ残基を表し；大文字はＲＮＡ残基を表し；ｄ＝２’‐デオキシ残基であり；「ｓ」はホスホロチオエート部を示す。 For example, according to the present invention, 2'-O-PivOM (Debert et al., Chem. Eur. J., 2008, 14, 9135), 2'-O-CEM (Ohgi et al., Org. Lett., 2005, 7, 7913). Wada et al., J. Org. Chem., 2012, 77, 7913), 2'-O-TOM (Pitch et al., Helv. Chem. Acta, 2001, 84, 3773), or 2'-O-TC (Dellinger). Et al., J. Am. Chem. Soc., 2011, 133, 11540), etc., using appropriate chiral RNA3'-phosphoramidite with appropriate 2'-OH protective groups to make multiple chiral phosphorothioate linkages into RNA oligonucleotides. Introduce within. Each of the following human PCSK9 siRNA sense strands with several chiral phosphorothioate nucleotide linkages and total chiral phosphorothioate nucleotide linkages can be prepared according to the present invention.

NOTE: Lowercase letters represent 2'-OMe RNA residues; uppercase letters represent RNA residues; d = 2'-deoxy residues; "s" indicates phosphorothioate moiety.

注記：小文字は２’‐ＯＭｅ ＲＮＡ残基を表し；大文字はＲＮＡ残基を表し；ｄ＝２’‐デオキシ残基であり；「ｓ」はホスホロチオエート部を示す。 Example of synthesis of human PCSK9 siRNA antisense strand having several chiral phosphorothioate nucleotide linkages and total chiral phosphorothioate nucleotide linkages.

NOTE: Lowercase letters represent 2'-OMe RNA residues; uppercase letters represent RNA residues; d = 2'-deoxy residues; "s" indicates phosphorothioate moiety.

Alternatively or additionally, the invention provides the preparation of several chiral phosphorothioate nucleotide linkages, and total chiral phosphorothioate nucleotide linkages, as well as siRNA double strands with a fully modified ribose moiety.

注記：小文字は２’‐ＯＭｅ ＲＮＡ残基を表し；大文字は２’‐Ｆ ＲＮＡ残基を表し；ｄ＝２’‐デオキシ残基であり；「ｓ」はホスホロチオエート部を示す。 For example, in certain embodiments, the corresponding desired ribose 2'-chemical modification, such as 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl (2'-OMe). Multiple chiral phosphorothioate linkages are introduced into the total ribose-modified RNA oligonucleotide using the appropriate chiral RNA3'-phosphoramidite having. To give a few examples, each of the following human PCSK9 siRNA fully modified 2'-F / 2'-OMe sense strands having several chiral phosphorothioate nucleotide linkages and total chiral phosphorothioate nucleotide linkages Can be prepared.

Note: Lowercase letters represent 2'-OMe RNA residues; uppercase letters represent 2'-F RNA residues; d = 2'-deoxy residues; "s" indicates phosphorothioate moiety.

注記：小文字は２’‐ＯＭｅ ＲＮＡ残基を表し；大文字は２’‐Ｆ ＲＮＡ残基を表し；ｄ＝２’‐デオキシ残基であり；「ｓ」はホスホロチオエート部を示す。 Example of synthesis of human PCSK9 siRNA fully modified 2'-F / 2'-OMe antisense strand having several chiral phosphorothioate nucleotide linkages and total chiral phosphorothioate nucleotide linkages.

Note: Lowercase letters represent 2'-OMe RNA residues; uppercase letters represent 2'-F RNA residues; d = 2'-deoxy residues; "s" indicates phosphorothioate moiety.

RNA chain thermal annealing and siRNA double strand preparation are performed to construct the double strand. Specifically, each RNA strand is mixed with the complementary RNA strand at an equimolar concentration of 10 μM in 1 × PBS. A total 0.5 mL solution is prepared for each double strand, the mixture is heated at 90 ° C. for 2 minutes and allowed to cool for several hours. The mixture is then stored at 4 ° C.

After the thermal RNA strand annealing step, all possible siRNA duplex combinations can be prepared by annealing any possible complementary strand of the antisense strand with any sense strand.

After transfection in HeLa cells or Hep3B cells (eg, as described herein), all siRNA double strands prepared can be evaluated in vitro for their PCSK9 gene silencing properties. In accordance with the present invention, different efficacy may be observed for different double strands, eg, the presence of any other chemical modification, which differs in number, position and / or conformation of chiral phosphorothioate backbone bonds, optionally one or more. Different in level and / or type.

For example, nuclease resistance, cellular penetration, endosome escape, double-stranded thermodynamic stability, double-stranded three-dimensional structure, affinity for various enzyme interaction mechanism steps, affinity for target mRNAs, specific off-target effects. Any or all siRNA properties, such as immunostimulation, duration of action, pharmacokinetics, etc., may be adjusted or affected by the stereochemistry of the chiral phosphorothioate backbone binding described above.

Example 64 Preparation of chiral-controlled oligonucleotide preparation

This example describes the preparation of a variety of specific chiral controlled compositions of the particular oligonucleotides described herein.

N ⁴ -Acetyl-5'-O-DMTr-2'-O-methylcytidine (1) (1.15 g, 1.91 mmol) was dissolved in anhydrous DCM (20 mL) and 2 equivalents of succinic anhydride (0. 383 g (3.82 mmol), and mixed with 3 equivalents of 4-N, N-dimethylaminopyridine (0.701 g, 5.73 mmol). The reaction was stirred under argon at room temperature. After the starting material has been completely consumed (1 hour) as determined by TLC, the solvent is evaporated to dry and the crude residue is dissolved in DCM containing 1% triethylamine and then 2%. Purification by flash silica gel chromatography using a 0-2% gradient of MeOH in DCM containing triethylamine. The yield of the high-purity compound (2) after evaporation was 1.50 g, 98%. MS (ESI + ve): Calculated value (M + H) ⁺ : 702.27, measured value: 702.34. N ⁴ -Acetyl-5'-O-DMTr-3'-O-Succinyl-2'-O-Methylcitidine (2) (0.18 g, 0.22 mmol) produced, N, N-diiropropylethylamine ( 0.18 ml, 0.79 mmol) and GE Custom Sport ™ Amino (1 g) were placed in DMF (5 mL), then HBTU (0.10 g, 0.26 mmol) was added. The mixture was shaken for 2 hours and then filtered. The carrier was washed with DMF, MeOH and finally DCM and then dried under vacuum. Trityl cation analysis (monitored at 504 nm) showed that the loading of nucleosides on carrier (3) was 180 μmol / g.

Example 65 Preparation of chirally controlled oligonucleotide preparation

Using a procedure similar to that described in Example 64, N4 ^- phenoxyacetyl-5'-O-DMTr-3'-O-succinyl-2'-O-methylguanosine (4, MS (ESI + ve)). : Calculated value (M + H) ⁺ : 834.30, measured value: 834.32) was filled in GE Custom Sport ™ Amino. Trityl cation analysis (monitored at 504 nm) showed that the loading of nucleosides on carrier (6) was 140 μmol / g.

In some examples, nucleotides containing sterically defined 2'-OMe phosphorothioate triester internucleotide linkages were administered on an ABI394 DNA / RNA synthesizer according to the cycle summarized in Table E-27 (60 mg). 5'-O-DMTr-2'-O-methyl- ^GPac (6, 140 μmol / g) or 5'-O-DMTr-2' bound with uncapped succinyl of 10.8 and 8.4 μmol, respectively) Synthesis was performed using a 10 μmol volume synthetic column packed with any of -O-methyl-C ^Ac (3, 180 μmol / g). The synthesis cycle was performed with a preliminary capping step (capping 2) and removal of the terminal 5'-O-DMTr group. The stereospecific sulfide step was performed after coupling of the corresponding chiral phosphoramide and a two-step capping step using a 0.3M methylthiosulfonic acid S- (2-cyanoethyl) reagent in ACN containing BSTFA. (Table E-27).

Once the automated oligonucleotide synthesis cycle was complete and the most 5'-O-DMTr groups were removed, the synthetic column was removed from the DNA / RNA synthesizer and dried under vacuum. Transfer the dried carrier to an empty glass manual peptide synthesizer and in a 10 mL solution 0.5 M 1,5-diazabicyclo (4.3.0) nona-5-ene (DBN), 0.25 MN, O in ACN. -Bis (trimethylsilyl) trifluoroacetamide (BSTFA) was continuously added to the carrier in a manual peptide synthesizer for 5 minutes without stopping the flow. The carrier was washed with ACN and dried under vacuum. The carrier was then treated with 5% EtOH / concentrated NH ₃ (20 mL) at 60 ° C. for 6 hours and left at room temperature for 12 hours. The carrier was removed by filtration and washed with concentrated NH ₃ . The filtrate was concentrated under vacuum and then purified by IEX.

Table E-27 Overview of oligonucleotide synthesis

Synthesis of oligonucleotide ONT-94: (All (Sp))-gsgsusgsgsasasgsgsgsc. t _R (IEX-HPLC): 18.26 minutes. Calculated MW: 3563.9; Measured MW: 3562.6.

Synthesis of oligonucleotide ONT-96: (All (Rp))-gsgsusgsgsasasgsgsgsc. t _R (IEX-HPLC): 18.16 minutes. Calculated MW: 3563.9; Measured MW: 3561.7.

Synthesis of oligonucleotide ONT-98: (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp)-gsgsusgsgsasasgsgsc. t _R (IEX-HPLC): 18.05 minutes. Calculated MW: 3563.9; Measured MW: 3562.5.

Synthesis of oligonucleotide ONT-100: (Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp)-gsgsusgsgsasagsgsc. t _R (IEX-HPLC): 17.86 minutes. Calculated MW: 3563.9; Measured MW: 3561.1.

Synthesis of oligonucleotide ONT-102: (Rp, Rp, Sp, Sp, Sp, Sp, Sp, Rp, Rp)-gsgsusgsgsasasgsgsc. t _R (IEX-HPLC): 18.30 minutes. Calculated MW: 3563.9; Measured MW: 3561.3.

Synthesis of oligonucleotide ONT-104: (Sp, Sp, Rp, Rp, Rp, Rp, Rp, Sp, Sp)-gsgsusgsgsasasgsgsc. t _R (IEX-HPLC): 17.95 minutes. Calculated MW 3563.9; Measured MW: 3562.7.

Synthesis of oligonucleotide ONT-95: (All (Sp))-gscscsusscscsag. t _R (IEX-HPLC): 14.78 minutes. Calculated MW: 2709.2; Measured MW: 2707.4.

Synthesis of oligonucleotide ONT-97: (All (Rp))-gscscsusscscsag. t _R (IEX-HPLC): 15.60 minutes. Calculated MW: 2709.2; Measured MW: 2708.3.

Synthesis of oligonucleotide ONT-99: (Rp, Sp, Rp, Sp, Rp, Sp, Rp)-gsscsusscscsasg. t _R (IEX-HPLC): 16.10 minutes. Calculated MW: 2709.2; Measured MW: 2708.0.

Synthesis of oligonucleotide ONT-101: (Sp, Rp, Sp, Rp, Sp, Rp, Sp)-gsscsusscscsasg. t _R (IEX-HPLC): 16.23 minutes. Calculated MW: 2709.2; Measured MW: 2708.2.

Synthesis of oligonucleotide ONT-103: (Rp, Rp, Sp, Sp, Sp, Rp, Rp)-gscscsusscsasg. t _R (IEX-HPLC): 16.26 minutes. Calculated MW: 2709.2; Measured MW: 2707.8.

Synthesis of oligonucleotide ONT-105: (Sp, Sp, Rp, Rp, Rp, Sp, Sp)-gscscsusscsasg. t _R (IEX-HPLC): 16.22 minutes. Calculated MW: 2709.2; Measured MW: 2710.0.

Oligonucleotides containing sterically defined chimeric 2'-OMe phosphorothioate triesters and 2'-O-MOE phosphorothioate triester internucleotide linkages are described in ABI394 DNA / RNA in the examples herein. It was synthesized by a method similar to the method.

Synthesis of oligonucleotide ONT-90: (All (Rp))-G _MOE sG _MOE susG _MOE sG _MOE sasasG _MOE sG _MOE sc. t _R (IEX-HPLC): 15.35 minutes. Calculated MW: 3828.2; Measured MW: 3826.5.

Synthesis of oligonucleotide ONT-119: (All (Sp))-G _MOE sG _MOE susG _MOE sG _MOE sasasG _MOE sG _MOE sc. t _R (IEX-HPLC): 16.42 minutes. Calculated MW: 3828.2; Measured MW: 3827.2.

Synthesis of oligonucleotide ONT-91: (All (Rp))-G _MOE scscsusscscassg. t _R (IEX-HPLC): 15.69 minutes. Calculated MW: 2753.3; Measured MW: 2751.5.

Synthesis of oligonucleotide ONT-120: (All (Sp))-G _MOE scscsusscscassg. t _R (IEX-HPLC): 14.71 minutes. Calculated MW: 2753.3; Measured MW: 2751.4.

Example 66 General IEX-HPLC method for analysis of crude and purified DMT Off RIPtide oligonucleotides:
Buffer A: 10 mM Tris HCl, 50% ACN, pH = 8.0
Buffer B: 10 mM Tris HCl, 800 mM NaClO ₄ , 50% ACN, pH = 8.0
Column: DIONEX, DNAPac, PA-100, for analysis, 4.0 x 250 mm
Column temperature = 60 ° C
Monitor signals at 254 and 280 nm Gradients used:

Example 67 Common UPLC-LCMS methods for analysis of purified DMT Off RIPtide oligonucleotides:
Buffer A: 15 mM TEA, 400 mM HFIP, water Buffer B: 50:50 Buffer A / Methanol Column: UPLC @ OST C ₁₈ 1.7 μm, 2.1 × 500 mm
Column temperature = 50 ° C
Gradient used:

Example 68 General IEX-HPLC Method for Purification of Crude DMT Off RIPtide Oligonucleotide Buffer A: 20 mM NaOH, pH = 11.0
Buffer B: 20 mM NaOH, 2.5 M NaCl, pH = 11.0
Column: Empty column Waters AP-2 (Waters), Source15Q carrier (GE Healthcare) was custom filled in-house. The same purified column was used for different sterically pure RIPtide oligonucleotides.
Equipment: AKTA refiner with P-900 pump, UPC-900 detector, and 50 mL injection SuperLoop (GE Healthcare) Buffer heater temperature setting = 70 ° C.
Column heater tape setting = 70 ° C
Monitor signal at 254 nm Fraction capacity: 5 mL
Gradient used:

The recovered fractions were individually analyzed by analytical IEX-HPLC using the above analytical conditions and gradients. High-purity fractions were collected to provide purified material with a purity of 95% or higher as determined by the UV absorption profile at 254 nm.

A common RP-Sep-Pak method for desalting purified RIPtide oligonucleotides. The stored pure fractional solution was loaded into a Sep-Pak cartridge (Waters, Sep-Pak Vac 35cc (10g) C ₁₈ cartridge) pre-prepared with water. After filling the sample (100 mL), the cartridge was washed with Milli-Q water (50 mL) to remove all salts and then washed with 50% ACN / water to elute full-length desalted RNA oligonucleotides. The recovered solution was concentrated under vacuum to a volume of 5 mL and lyophilized from water.

A panel of sterically controlled total phosphorothioate-modified RIPtides (eight or tetramers) that bind to bare hTR will be examined for in vitro inhibition of the activity of the telomerase RNP complex. Previously using HeLa cell extract using the Cy5-TRAP assay (Shay et al., Nat. Protoc., 2006, 1, 1583), a variant of TRAP (Shay et al., Science, 1994, 266, 2011). According to the protocol reported in (Verdine et al., J. Biol. Chem., 2012, 287, 18843), the IC ₅₀ value of the sterically controlled phosphorothioate RIPtide is determined.

The TRAP assay is performed with some modifications according to the previously reported protocol using fluorescence as a quantitative system. Briefly, the fluorescent artificial substrate is extended with telomerase at 30 ° C. for 30 minutes and then amplified in 30 PCR cycles (34 ° C. 30 seconds, 59 ° C. 30 seconds, 72 ° C. 1 minute). In replication experiments, the inhibitory activity of RIPtide is initially assessed in HeLa cell extracts and uses a concentration range of 600 pM to 60 μM. Experiments with selected RIPtides are repeated in Hela, DU145 (prostate cancer) and HEK293 cell extracts in a concentration range of 0.06 pM-60 μM. Several controls are used in these assays: positive controls (untreated cell solubilized products), negative controls (buffer only, heat-inactivated, RNase-treated cell extracts) and PCR amplification. Control (60 μM RIPtide added after telomerase extension and before PCR). In certain embodiments, chiral-controlled RIPtide agents show increased inhibition of hTR compared to positive controls and / or increased inhibition of hTR compared to negative controls; some. In one embodiment of the above, some, but optionally not all, of a given sequence of RIPtides exhibit such properties. In some embodiments, "increasing" in this context means increasing about 5 to about 10 times. In some embodiments, "increasing" means about 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5. It means 5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 times or more.

Cell culture conditions. Transformed embryonic kidney cell line HEK293 and prostate cancer cell line DU145 are maintained in 5% CO ₂ at 37 ° C. in DMEM supplemented with 10% fetal bovine serum. Soluble cell extracts for TRAP assay are prepared by detergent lysis of 106 cells with 200 μL of 1 × CHAPS Lysis buffer (Chemicon) as described in the manufacturer's instructions.

According to the present invention, different efficacy and / or different other properties may be observed for all three-dimensional controlled phosphorothioate RIPtides of various conformations. For example, RIPtide characteristics such as nuclease resistance, tissue accumulation, cellular penetration, endosome escape, three-dimensional structure of RIPtide, affinity for target folding hTR RNA, immune stimulation, duration of action, pharmacokinetics, etc. share the same sequence. However, they may differ between different positions of one or more chiral phosphorothioate backbone bonds and / or different chiral-controlled RIPtide agents with respect to steric science.

Example 69: Chiral-controlled oligonucleotide compositions exhibit different activities in vivo as compared to chiral-uncontrolled compositions having the same sequence.

In this example, the in vivo pharmacological activity of a chirally pure oligonucleotide is expressed in an "original" sterically random mixture (ie, the same sequence as the chirally pure oligonucleotide, but not as chirally pure. No, eg, a composition containing the resulting oligonucleotide prepared by a sterically random process) compared to the activity observed. Pure oligonucleotides are synthesized and dispensed as four chirals, each with a sequence complementary to the sequence of the gene encoding a particular target transcript or protein of interest, each at two dosing levels, 2 per week. It was administered to animals expressing the target gene for 5 weeks. The level of encoded protein was quantified. In this example, an oligonucleotide having an antisense sequence on human apolipoprotein-B (ApoB) (hence targeting human apolipoprotein-B (ApoB)) is used to demonstrate the concept of transgenic mouse-expressing human ApoB. Used for.

Test product The test product is PBS alone (ie, without oligonucleotide control) or a related oligonucleotide composition (ie, mipomersen (ONT-41), ONT-75, ONT-77, ONT-80 or ONT-81 oligonucleotide (structure). See Example 52 for)) in 1 x PBS (diluted from 10 x PBS (Life Technologies, AM9624) using nuclease-free water (Qiagen, 129115)) at 5 and 10 mg. Formulated at 0.5 and 1 mg / mL, respectively, for administration at / kg. The formulation was based on absolute mass without adjustment of the active substance. Oligonucleotide concentrations were confirmed by measurement with Carry-100 UV-Vis (Agilent Technologies). Samples of all test items were examined for endotoxin levels using the Dynamics Pyrochrome dye-producing endotoxin assay (Associates of Cape Cod, 1500-5, E005-5). All test samples were found to have an amount of endotoxin below the tolerance of 0.5 EU / ml.

Animal and In vivo Procedures Taconic (huApoB mice) of female transgenic mice expressing high plasma concentrations of human apolipoprotein B100 and lipoprotein (a) (J. Clin. Invest. 92: 3029-3037) Strain B6. SJL-Tg (APOB) l 102Sgy N20 + ?, model # 1004-F). All animals were genetically determined prior to delivery.

Mice were delivered and were acclimatized for at least 7 days prior to the start of the study. All mice were given the usual solid diet and water freely and were not fasted prior to compound administration. Mice were randomly assigned to study groups and at 10 ml / kg based on the body weight of individual mice measured prior to administration on each dosing day, first, fourth, eighth, 11, 15, 18, 22, 25, 29 and 32. Intraperitoneal administration (IP) was performed on the same day. No. 0 (the day before the first administration),
During the 17th, 24th, 31st, 38th, 45th, 52nd and 60th days of investigation, blood was collected by submandibular (cheek) hemorrhage, sacrificed by heart puncture on day 66 and then treated with serum.

Apolipoprotein B Protein Assay ApoB protein levels in serum were measured using the ApoB Human ELISA Kit (Abcam, ab108807). Serum samples were diluted 1: 20,000 and assayed unmodified according to the kit recommended protocol. Automated cleaning was performed using Aquamax 4000 (Molecular Devices) with a 30 second immersion cycle. After incubation with the dye source substrate for 12 minutes, the reaction was stopped, and measurement was performed with Spectramax M5 (Molecular Devices).

A standard curve was created by plotting the standard concentration on the x-axis and the corresponding mean 450 nm absorbance on the y-axis. The optimal line was determined by regression analysis using log-log curve fitting. Each assay plate contained negative (PBS-treated) and positive (mipomersen-treated) controls, and each mouse serum sample was assayed with four-technique replication.

In each sample, the mean absolute level of ApoB was normalized to the mean of the PBS-treated group to obtain the relative level of ApoB protein expression. In one example, animals were excluded from the analysis because the measurements deviated from the cohort mean by 2 standard deviations. Relative levels of ApoB protein expression were used for statistical analysis by dual-arranged ANOVA followed by Newman-Keuls post-test (Graphpad Prism).

ａ：ミポメルセン対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０５（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｂ：ミポメルセン対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｃ：ミポメルセン対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．００１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｄ：ミポメルセン対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０００１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｗ：ＰＢＳ対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．５（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｘ：ＰＢＳ対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｙ：ＰＢＳ対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．００１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｚ：ＰＢＳ対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０００１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験） Results Results are shown in Table E-28a and FIG. 40. Compared with PBS, 5 mg / kg IP ONT-75 resulted in a significant reduction in serum ApoB protein levels to 87% (p <0.05) on day 24 of the study. In 5 mg / kg IP ONT-77 compared to PBS, serum ApoB protein levels on days 24 and 31 of the study were 72% (p <0.0001) and 81% (p <0.) Of the control levels, respectively. The result was a significant decrease to 05). At 5 mg / kg IP ONT-80 compared to PBS, serum ApoB protein levels on days 24 and 31 of the study were 83% (p <0.05) and 80% (p <0.) Of control levels, respectively. The result was a significant decrease to 05). Compared to PBS, with 5 mg / kg IP ONT-81, serum ApoB protein levels on days 17 and 24 of the study were 85% (p <0.05) and 70% (p <0.) Of the control levels, respectively. The result was a significant decrease to 0001).

Table E-28a: Serum human apolipoprotein B levels for PBS after multiple IP administration of 5 mg / kg stereoisomer or mipomersen in huApoB mice (N = 4-5)

a: Statistically different from the Mipomersen control group (ONT-41), p <0.05 (two-way ANOVA and Newman-Keuls post-test)
b: Statistically different from the Mipomersen control group (ONT-41), p <0.01 (two-way ANOVA and Newman-Keuls post-test)
c: Statistically different from the Mipomersen control group (ONT-41), p <0.001 (two-way ANOVA and Newman-Keuls post-test)
d: Statistically different from the Mipomersen control group (ONT-41), p <0.0001 (two-way ANOVA and Newman-Keuls post-test)
w: Statistically different from the PBS control group (ONT-41), p <0.5 (two-way ANOVA and Newman-Keuls post-test)
x: Statistically different from the PBS control group (ONT-41), p <0.01 (two-way ANOVA and Newman-Keuls post-test)
y: Statistically different from the PBS control group (ONT-41), p <0.001 (two-way ANOVA and Newman-Keuls post-test)
z: Statistically different from the PBS control group (ONT-41), p <0.0001 (two-way ANOVA and Newman-Keuls post-test)

For ONT-75 administered in the same dosing paradigm as compared to 5 mg / kg IP mipomersen, at surveys 17, 24, 31 and 38 (p <0.0001) and 24 (p <0.05). Serum ApoB protein levels were significantly reduced. For ONT-77 administered in the same dosing paradigm as compared to 5 mg / kg IP mipomersen, surveys 17, 31 and 38 (p <0.0001) 24 days (p <0.001) and 45 days (p <0.001). Serum ApoB protein levels at p <0.05) were significantly reduced. Serum ApoB protein levels at study days 17, 24, 31 and 38 (p <0.0001) were significantly reduced in ONT-80 administered in the same dosing paradigm compared to 5 mg / kg IP mipomersen. However, there was no difference on the 45th day (p> 0.05). For ONT-81 administered in the same dosing paradigm compared to 5 mg / kg IP mipomersen, studies 17th, 31st and 38th (p <0.0001) and 24th (p <0.001) and 45th. Serum ApoB protein levels at (p <0.01) were significantly reduced. FIG. 40 shows the time course of serum human apolipoprotein B levels for PBS after IP administration of 5 mg / kg stereoisomer or mipomersen in huApoB mice. The down arrow indicates the number of days of administration. The group shows a normalized group mean to the PBS control group, and each group consists of 4-5 animals. Error bars represent standard deviation.

The results of the 10 mg / kg dosing paradigm are shown in Table E-28b and FIG. 53. With respect to PBS, at 10 mg / kg IP ONT-75, serum ApoB protein levels at study days 17, 24 and 38 were 78% (p <0.0001), 76% (p <0.0001) and 82%, respectively. The result was a significant decrease to (p <0.001). At 10 mg / kg IP ONT-77 compared to PBS, serum ApoB protein levels at study days 17, 24, 31, 38, 45 and 52 were 62% (p <0.0001) of control levels, respectively. To 61% (p <0.0001), 54% (p <0.0001), 59% (p <0.0001), 72% (p <0.0001) and 73% (p <0.0001) The result was a significant decrease. At 10 mg / kg IP ONT-80 compared to PBS, serum ApoB protein levels at study days 17, 24, 31, 38, 45, 52 and 66 were 55% (p <0.0001) of control levels, respectively. ), 59% (p <0.0001), 73% (p <0.0001), 67% (p <0.0001), 76% (p <0.0001), 76% (p <0.0001). ) And 80% (p <0.01). At 10 mg / kg IP ONT-81 compared to PBS, serum ApoB protein levels at study days 17, 24, 31, 38 and 45 were 49% (p <0.0001) and 62% of control levels, respectively. The results were significantly reduced to (p <0.0001), 71% (p <0.0001), 74% (p <0.0001) and 79% (p <0.001).

ａ：ミポメルセン対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０５（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｂ：ミポメルセン対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｃ：ミポメルセン対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．００１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｄ：ミポメルセン対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０００１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｗ：ＰＢＳ対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０５（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｘ：ＰＢＳ対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｙ：ＰＢＳ対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．００１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｚ：ＰＢＳ対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０００１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験） Table E-28b: Serum human apolipoprotein for PBS after multiple IP administration of 10 mg / kg stereoisomer (ONT-75, -77, -80 or -81) or mipomersen in huApoB mice (N = 4-5). B level

a: Statistically different from the Mipomersen control group (ONT-41), p <0.05 (two-way ANOVA and Newman-Keuls post-test)
b: Statistically different from the Mipomersen control group (ONT-41), p <0.01 (two-way ANOVA and Newman-Keuls post-test)
c: Statistically different from the Mipomersen control group (ONT-41), p <0.001 (two-way ANOVA and Newman-Keuls post-test)
d: Statistically different from the Mipomersen control group (ONT-41), p <0.0001 (two-way ANOVA and Newman-Keuls post-test)
w: Statistically different from the PBS control group (ONT-41), p <0.05 (two-way ANOVA and Newman-Keuls post-test)
x: Statistically different from the PBS control group (ONT-41), p <0.01 (two-way ANOVA and Newman-Keuls post-test)
y: Statistically different from the PBS control group (ONT-41), p <0.001 (two-way ANOVA and Newman-Keuls post-test)
z: Statistically different from the PBS control group (ONT-41), p <0.0001 (two-way ANOVA and Newman-Keuls post-test)

For ONT-75 administered in the same dosing paradigm as compared to 10 mg / kg IP mipomersen, studies 17, 24, 31, 38, 45, 52, 60 days (p <0.0001) and 66 days (p <0.0001). Serum ApoB protein levels at <0.001) showed a significantly lower reduction (knockdown). For ONT-77 administered in the same dosing paradigm compared to 10 mg / kg IP mipomersen, surveys 17, 24, 31, 38 and 45 days (p <0.0001), 60 days (p <0.05). ) And serum ApoB protein levels at day 66 (p <0.01) showed a significantly lower reduction (knockdown). For ONT-80 administered in the same dosing paradigm compared to 10 mg / kg IP mipomersen, studies 24th, 31st, 38th and 45th (p <0.0001), 17th (p <0.001) and Serum ApoB protein levels at day 66 (p <0.01) showed a significantly lower reduction (knockdown). For ONT-81 administered in the same dosing paradigm compared to 10 mg / kg IP mipomersen, studies 24th, 31st, 38th and 45th (p <0.0001), 17th (p <0.01), Serum ApoB protein levels at days 52 and 66 (p <0.001) showed a significantly less reduction (knockdown).

By day 38 (ie, 6 days after the last dose), serum ApoB protein levels had returned to baseline after ONT-75. Considering the decrease in serum ApoB protein levels at the start, the rate of recovery of serum ApoB protein levels to baseline was slower than after ONT-77 and ONT-80, and serum ApoB protein levels were with Mipomersen levels by day 52. It was similar.

The results of the 5 mg / kg dosing paradigm with a further sterically pure structure are shown in Table E-28c and FIG. 54. In 5 mg / kg IP ONT-87 compared to PBS, serum ApoB protein levels at study days 17, 24, 31 and 38 were 27% (p <0.0001) and 40% (p) of control levels, respectively. The results were significantly reduced to <0.0001), 55% (p <0.0001) and 71% (p <0.0001). In 5 mg / kg IP ONT-88 compared to PBS, serum ApoB protein levels at study days 17, 24, 31 and 38 were 47% (p <0.0001) and 34% (p) of control levels, respectively. The results were significantly reduced to <0.0001), 69% (p <0.0001) and 85% (p <0.0001). In 5 mg / kg IP ONT-89 compared to PBS, serum ApoB protein levels at study days 17, 24 and 31 were 43% (p <0.0001) and 48% (p <0) of control levels, respectively. The result was a significant decrease to .0001) and 85% (p <0.05).

ａ：ミポメルセン対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０５（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｂ：ミポメルセン対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｃ：ミポメルセン対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．００１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｄ：ミポメルセン対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０００１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｗ：ＰＢＳ対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０５（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｘ：ＰＢＳ対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｙ：ＰＢＳ対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．００１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｚ：ＰＢＳ対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０００１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験） Table E-28c: Serum human apolipoprotein B levels for PBS after multiple IP administration of 5 mg / kg stereoisomer (ONT-87, -88, or -89) or mipomersen in huApoB mice (N = 3-4).

a: Statistically different from the Mipomersen control group (ONT-41), p <0.05 (two-way ANOVA and Newman-Keuls post-test)
b: Statistically different from the Mipomersen control group (ONT-41), p <0.01 (two-way ANOVA and Newman-Keuls post-test)
c: Statistically different from the Mipomersen control group (ONT-41), p <0.001 (two-way ANOVA and Newman-Keuls post-test)
d: Statistically different from the Mipomersen control group (ONT-41), p <0.0001 (two-way ANOVA and Newman-Keuls post-test)
w: Statistically different from the PBS control group (ONT-41), p <0.05 (two-way ANOVA and Newman-Keuls post-test)
x: Statistically different from the PBS control group (ONT-41), p <0.01 (two-way ANOVA and Newman-Keuls post-test)
y: Statistically different from the PBS control group (ONT-41), p <0.001 (two-way ANOVA and Newman-Keuls post-test)
z: Statistically different from the PBS control group (ONT-41), p <0.0001 (two-way ANOVA and Newman-Keuls post-test)

On T-87 administered in the same dosing paradigm compared to 5 mg / kg IP mipomersen, serum ApoB protein levels at day 31 of study (p <0.01) showed a significantly higher reduction (knockdown). rice field. Serum ApoB protein levels on day 17 of study (p <0.05 of study) were significantly lower (knockdown) in ONT-88 administered in the same dosing paradigm compared to 5 mg / kg IP mipomersen. showed that. Serum ApoB protein levels at study day 38 (p <0.05) showed a significantly lower reduction (knockdown) in ONT-89 administered in the same dosing paradigm compared to 5 mg / kg IP mipomersen. rice field.

ａ：ミポメルセン対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０５（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｂ：ミポメルセン対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｃ：ミポメルセン対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．００１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｄ：ミポメルセン対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０００１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｗ：ＰＢＳ対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０５（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｘ：ＰＢＳ対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｙ：ＰＢＳ対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．００１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験）
ｚ：ＰＢＳ対照群（ＯＮＴ‐４１）と統計的に異なる、ｐ＜０．０００１（二元配置ＡＮＯＶＡとＮｅｗｍａｎ－Ｋｅｕｌｓ事後試験） The results of the 10 mg / kg dosing paradigm with a further sterically pure structure are shown in Table E-28d and FIGS. 55 and 56. At 10 mg / kg IP ONT-87 compared to PBS, serum ApoB protein levels at study 17 and 24 were 27% (p <0.0001) and 14% (p <0.0001) of control levels, respectively. ) Was significantly reduced. At 10 mg / kg IP ONT-88 compared to PBS, serum ApoB protein levels at study days 17 and 24 were 23% (p <0.0001) and 17% (p <0. The result was a significant decrease to 0001). At 10 mg / kg IP ONT-89 compared to PBS, serum ApoB protein levels at study days 17 and 24 were 29% (p <0.0001) and 23% (p <0. The result was a significant decrease to 0001).

Table E-28d: Serum human apolipoprotein B levels for PBS after multiple IP administration of 10 mg / kg stereoisomer (ONT-87, -88, or -89) or mipomersen in huApoB mice (N = 3-4).

a: Statistically different from the Mipomersen control group (ONT-41), p <0.05 (two-way ANOVA and Newman-Keuls post-test)
b: Statistically different from the Mipomersen control group (ONT-41), p <0.01 (two-way ANOVA and Newman-Keuls post-test)
c: Statistically different from the Mipomersen control group (ONT-41), p <0.001 (two-way ANOVA and Newman-Keuls post-test)
d: Statistically different from the Mipomersen control group (ONT-41), p <0.0001 (two-way ANOVA and Newman-Keuls post-test)
w: Statistically different from the PBS control group (ONT-41), p <0.05 (two-way ANOVA and Newman-Keuls post-test)
x: Statistically different from the PBS control group (ONT-41), p <0.01 (two-way ANOVA and Newman-Keuls post-test)
y: Statistically different from the PBS control group (ONT-41), p <0.001 (two-way ANOVA and Newman-Keuls post-test)
z: Statistically different from the PBS control group (ONT-41), p <0.0001 (two-way ANOVA and Newman-Keuls post-test)

On T-87 administered in the same dosing paradigm as compared to 10 mg / kg IP mipomersen, serum ApoB protein levels at study day 17 (p <0.05) showed a significantly higher reduction (knockdown). rice field. On T-88 administered in the same dosing paradigm as compared to 10 mg / kg IP mipomersen, serum ApoB protein levels at study day 17 (p <0.05) showed a significantly higher reduction (knockdown). rice field. Serum ApoB protein levels on day 17 (p <0.05) of study showed a significantly higher reduction (knockdown) in ONT-89 administered in the same dosing paradigm compared to 10 mg / kg IP mipomersen. rice field.

Thus, in at least some embodiments, the invention exhibits biological activity and appropriate references (eg, the same sequence but different chiral specificities, including particularly sterically random agents). To provide a chiral-controlled oligonucleotide composition characterized by a slower rate of decay of its activity over time and / or with the passage of time as compared to an oligonucleotide agent. In some embodiments where such long-lasting and / or slow decay rates are observed, the provided chiral-controlled composition can be administered at a smaller total dose and / or over a longer period of time. Between two or more doses used with a sterically random composition, they may be administered according to a dosing regimen to achieve comparable biological and / or pharmacological effects. For example, as demonstrated in this example, the slow recovery of serum ApoB proteins to baseline after treatment with a chiral-pure oligonucleotide as compared to mipomersen is due to the chiral-pure ApoB oligonucleotide. It suggests that it can be administered less frequently than a sterically random mixture of (eg, mipomersen).

The results shown in this example show that, for example, an oligonucleotide composition pure as chiral is a suitable reference (eg, an oligonucleotide of the same sequence but different chiral specificity, particularly comprising a sterically random agent. Drugs), in detail, demonstrate that they can have significantly different pharmacological activities compared to the "original" sterically random drug. Those skilled in the art will appreciate that the chiral controlled oligonucleotide compositions provided by the present disclosure have unexpected activity and properties in view of this demonstration. Those skilled in the art will be provided with a variety of techniques, including therapeutic methods using a different dosing regimen than those used for non-chiral control compositions (eg, those having the same nucleotide sequence) in view of the present disclosure. You will understand more about that. In some embodiments, individual doses of relatively high chiral controls (eg, pure as chiral) may be used compared to controls (eg, sterically random controls). In some embodiments, individual doses of relatively less chiral controls (eg, pure as chiral) may be used compared to controls (eg, sterically random controls). In some embodiments, administration of a relatively infrequent chiral control (eg, pure as chiral) oligonucleotide is administered within a given time period as compared to a control (eg, a sterically random control). You may use it.

Example 70: siRNA agents containing chiral controlled oligonucleotides inhibit PCSK-9

This example demonstrates the success of inhibition of target gene expression using siRNA agents consisting of chirally controlled oligonucleotides described herein. In particular, this example describes hybridization of individual oligonucleotide chains prepared by the chiral-controlled synthesis described herein, providing a double-stranded chiral-controlled siRNA oligonucleotide composition. This example further demonstrates the success of cell transfection with this agent and further demonstrates the success of inhibition of target gene expression.

SiRNA transfection of chiral siRNA molecules Hep3B or HeLa cells were backtransfected in 96-well plates at a density of 2.0 × 10 ⁴ cells / well. Transfection of siRNA was performed using lipofectamine RNAiMax (Life Technologies, Catalog No. 13778-150) and the manufacturer's protocol, reducing the amount of lipofectamine RNAiMax to 0.2 μl per well. Starting from 1 μM, 12 1: 3 siRNA double-stranded diluents were prepared. 10 μl of 10 × siRNA double strands were then lipoplexed with 9.8 μl of serum-free medium and a prepared mixture of 0.2 μl of lipofectamine RNAiMax per well. After 10-15 minutes of incubation, 2.0 × 10 ⁴ cells / well in 80 μl EMEM cell growth medium (ATCC, 30-2003) was added to a final volume of 100 μl per well. Two separate transfection events were performed for each dose.

Twenty-four hours after transfection, Hep3B or HeLa cells were lysed and PCSK9 mRNA was purified using the MagMAX ™ -96 Total RNA Isolation Kit (Life Technologies, AM1830); high volume with RNase inhibitor. 15 μl of cDNA was synthesized with a cDNA reverse transcription kit (Life Technologies, 4374967). FAM-labeled Taqman probe set for PCSK9 (Life Technologies, Hs03037355_m1) and VIC-labeled GAPDH primer limited endogenous control (Life, 04 707 494 001) using Probes Master Mix (Roche, 04 707 494 001) according to the manufacturer's protocol. Using 3.3), gene expression was evaluated by real-time PCR on Lightcycller 480 (Roche).

表Ｅ‐３０ 相対ＩＣ５０値［ＨｅＬａトランスフェクション］

表Ｅ‐３１ ３ホスホロチオエート立体中心を有するｓｉＲＮＡの相対ＩＣ５０値［ＨｅＬａトランスフェクション］

Values were calculated using IC50 and data analysis delta delta Ct method. Samples were normalized to hGAPDH and calibrated to fake transfected and untreated samples. A sterically random molecule was used as a positive control. The Graphpad prism was used to represent the data as the mean of two biological replications. To calculate the relative IC50, a four-parameter linear regression curve was fitted to the data and the bottom and top were constrained to constants 0 and 100, respectively.

Table E-29 Relative IC50 values [Hep3B transfection]

Table E-30 Relative IC50 values [HeLa transfection]

Table E-31 3 Relative IC50 values of siRNA with a phosphorothioate stereocenter [HeLa transfection]

Figures 45-50 show the results of these investigations. FIG. 45 shows the residual% PCSK-9 mRNA after Hep3B treatment with siRNA double strand. FIG. 46 shows the residual% PCSK-9 mRNA curve fitting after Hep3B treatment with siRNA double strands. FIG. 47 shows the residual% PCSK-9 mRNA after HeLa treatment with siRNA double strand. FIG. 48 shows the residual% PCSK-9 mRNA curve fitting after HeLa treatment with siRNA double strands. FIG. 49 shows the residual% PCSK-9 mRNA after HeLa treatment with a siRNA double strand containing 3 phosphorothioate stereocenters. FIG. 50 shows the residual% PCSK-9 mRNA curve fitting after HeLa treatment with siRNA double strands containing 3 phosphorothioate stereocenters.

As can be seen, in molecules with a single stereochemically defined phosphorothioate in each strand, it is observed that the efficacy is slightly increased in molecules with SP _{stereochemistry} in phosphorothioate in both the sense and antisense strands. Was done. Although the siRNA agent illustrated in detail contains only one chiral center per chain, those skilled in the art reading this disclosure have demonstrated differences as proof of the principle that the presence of chiral affects activity. You will recognize the importance of the effect.

The chiral effect is more pronounced in siRNAs with more than 2 stereocenters. For siRNAs with 3 chiral phosphorothioates (one in the sense strand and two in the antisense strand), combined with _RP stereochemistry at the 3'end of the antisense strand (between nucleotides n20 and n21) 5 'SP _{stereochemistry} at the ends (between nucleotides n1 and n2) had a detrimental effect on efficacy. However, the _RP stereochemistry at the 5'end (between nucleotides n1 and n2) combined with the SP stereochemistry at the 3'end (between nucleotides _n20 and n21) of the antisense strand is a sterically random control siRNA. In contrast, a significant improvement in PCSK-9 mRNA knockdown was shown. This result suggests that both chains are affected by the stereochemistry of phosphorothioates. The observed difference in efficacy appears to be increased with drugs with more chiral centers, and in addition, the location and type of chiral center becomes active through, for example, stability, efficacy, and / or both. Seems to be able to influence. Accordingly, one of ordinary skill in the art reading the present disclosure will teach the composition of both single-stranded and double-stranded chiral-controlled oligonucleotides, and in at least some embodiments such chiral-controlled oligonucleotides and agents. It will be appreciated that the present disclosure provides its use in identifying sterically random agents having the same sequence.
Example 71 Further exemplary oligonucleotides and their synthesis

Oligonucleotides N101-N104 were oxalylated to a 1 μmol synthetic column and 1.7 μmol dG bound to HCP according to the synthetic cycle summarized in Table E-32 using automated synthesis in the ABI-394 DNA / RNA synthesizer. It was synthesized using ^{CE and Pac} . The synthesis cycle was carried out with the terminal 5'-O-DMTr group removed (DMT Off). After completion of automated oligonucleotide synthesis, the HCP carrier was washed with dehydrated ACN and dried under vacuum. The dried HCP was placed in a plastic vial and treated with 1 mL of saturated NH ₃ / Py at 55 ° C. for 1 hour and then with 1 mL of dehydrated propylamine (ratio 1: 4) in dehydrated pyridine for 18 hours at room temperature. .. The solvent was then evaporated and the residue was resuspended in HCl aqueous solution containing 10% DMSO at pH about 1.5 and the HCP carrier was filtered off. The crude product was purified by reverse phase preparative HPLC (following the procedure below). Fractions with a purity greater than 95% were collected, concentrated and desalted by reverse phase HPLC (following the procedure below). The final desalting product was lyophilized from water.
Table E-32 Outline of oligonucleotide synthesis in the DNA / RNA synthesizer ABI-394 used for the synthesis of oligonucleotides N101-N102.

Example 72 HPLC method of N101 and N102:
Buffer A: 50 mM TEAA, pH = 9.6 (adjusted with TEA)
Buffer B: ACN
Column: XBridge C ₈ , 3.5 μm, 4.6 × 50 mm, product number # 186003034
Buffer solution heater set temperature = 35 ° C
Monitor signals at 254 and 280 nm Gradients used:

Example 73 General UPLC-LCMS Methods for N101 and N102:
Buffer A: 10 mM ammonium formate, pH 9.5 (adjusted with NH ₃ aq.)
Buffer B: ACN
Column: UPLC @ OST C ₁₈ 1.7 μm, 2.1 × 500 mm
Column temperature = 35 ° C
Gradient used:

１００μＬの４９～５１％リン酸を各分画（ｐＨ３）に加え、ＨＰＬＣにより確認し、次に凍るまで－８０℃で保管した。分画を１／２の体積になるまで凍結乾燥機で２～３時間保管し、その後－８０℃で保管した。
Examples 74 General Purification Methods for N101 and N102:
Buffer A: 50 mM TEAA, pH = 9.6 (adjusted with TEA)
Buffer B: ACN
Column: XBridge Prep C ₁₈ , 5 μm, C ₁₈ , 250 × 10 mm, part number 186003256
Buffer heater set temperature = room temperature Monitor signal at 254 and 280 nm Gradient used:

100 μL of 49-51% phosphoric acid was added to each fraction (pH 3), confirmed by HPLC and then stored at −80 ° C. until freezing. Fractions were stored in a lyophilizer for 2-3 hours until the volume was reduced to 1/2, then stored at -80 ° C.

回収した分画を凍るまで－８０℃で保管し、乾燥するまで凍結乾燥機で保管し、ＨＰＬＣにより確認した。 Example 75 General Desalting Methods for N101 and N102:
Buffer A: 10 mM formic acid Buffer B: ACN
Column: XBridge Prep C ₁₈ , 5 μm, 250 × 10 mm, product number # 186003256
Buffer heater set temperature = room temperature Monitor signal at 254 and 280 nm Gradient used:

The collected fraction was stored at −80 ° C. until frozen, stored in a lyophilizer until dried, and confirmed by HPLC.

Example 76 Synthesis of oligonucleotide N101 ((All- (Rp) -d [5mCs16As16Gs16T]) (s16 is as shown below)

Oligonucleotide N101 was synthesized and purified as described above. RT in RP-HPLC (HPLC method N1): 15.9 minutes. UPLC / ESI-MS: Calculated value: 1614.64; Measured value [+ H]: 165.06.

Example 77 Synthesis of oligonucleotide N102 (All- (Sp) -d [5mCs16As16Gs16T])

Oligonucleotide N102 was synthesized and purified as described above. RT in RP-HPLC (HPLC method N1): 16.4 minutes. UPLC / ESI-MS: Calculated value: 1614.64; Measured value [+ H]: 1614.97.

Example 78 Synthesis of oligonucleotide N103 (All- (Rp) -d [5mCs16As16Gs16Ts165mCs16Ts16Gs165mCs16Ts16Ts165mCs16G])

Oligonucleotide N103 was synthesized as described above. RT in RP-HPLC (HPLC method N1): 18.4 minutes. UPLC / ESI-MS: C ₁₉₇ H ₃₁₁ N ₆₂ O ₆₂ P ₁₁ S ₁₁ Calculated value: 5233.38; Measured value: 5234.7.

Example 79 Synthesis of Oligonucleotide N104 (All- (Sp) -d [5mCs16As16Gs16Ts165mCs16Ts16Gs165mCs16Ts16Ts165mCs16G])

Oligonucleotide N104 was synthesized as described above. RT in RP-HPLC (HPLC method N1): 18.7 minutes. UPLC / ESI-MS: Calculated value: 5233.38; Measured value: 5232.9.

Oligonucleotides N105-N106 were oxalylated to a 1 μmol synthetic column and 1.7 μmol dG bound to HCP according to the synthetic cycle summarized in Table E-33 using automated synthesis in the ABI-394 DNA / RNA synthesizer. It was synthesized using ^{CE and Pac} . The synthesis cycle was carried out with the terminal 5'-O-DMTr group removed (DMT Off). After completion of automated oligonucleotide synthesis, the HCP carrier was washed with dehydrated ACN and dried under vacuum. The dried HCP was placed in a plastic vial and treated with 1 mL of saturated NH ₃ / i-PrOH at 55 ° C. for 16 hours. The solvent was then evaporated and the residue was resuspended in a 10% DMSO-containing aqueous solution at pH 7.0 and the HCP carrier was filtered off. The crude product is purified by reverse phase preparative HPLC. Fractions with a purity greater than 95% are collected, concentrated and desalted by reverse phase HPLC. The final desalting product is lyophilized from water.

Table E-33 Outline of oligonucleotide synthesis in the DNA / RNA synthesizer ABI-394 used for the synthesis of oligonucleotide N105-N106.

Example 80 HPLC Method for N101 and N102 Buffer A: 50 mM TEAA, pH = 7.0
Buffer B: ACN
Column: XBridge C ₈ , 3.5 μm, 4.6 × 150 mm, product number # 186003034
Buffer solution heater set temperature = 35 ° C
Monitor signals at 254 and 280 nm Gradients used:

Example 81 General UPLC-LCMS methods for N101 and N102:
Buffer A: 10 mM ammonium formate, pH 7.0
Buffer B: ACN
Column: UPLC @ OST C ₁₈ 1.7 μm, 2.1 × 500 mm
Column temperature = 35 ° C
Gradient used:

Example 82 Synthesis of oligonucleotide N105 (All- (Rp) -d [5mCs9As9Gs9T] (s9 is as shown below))

Oligonucleotide N105 was synthesized as described above. RT in RP-HPLC (HPLC method N2): 14.1 minutes. UPLC / ESI-MS: Calculated value: 1977.78; Measured value [-H]: 1978.8.

Example 83 Synthesis of oligonucleotide N106 (All- (Sp) -d [5mCs9As9Gs9T])

Oligonucleotide N106 was synthesized and purified as described above. RT in RP-HPLC (HPLC method N2): 14.7 minutes. UPLC / ESI-MS: Calculated value: 1977.78; Measured value [-H]: 1977.37.

Oligonucleotide xxx was oxalylated to a 1 μmol synthetic column and 1.7 μmol dG ^CE, according to the synthetic cycle summarized in Table E-33, using automated synthesis in the ABI-394 DNA / RNA synthesizer. Synthesized using ^Pac . The synthesis cycle was carried out with the terminal 5'-O-DMTr group removed (DMT Off). After completion of automated oligonucleotide synthesis, the HCP carrier was washed with dehydrated ACN and dried under vacuum. The dried HCP was placed in a plastic vial and treated with 1 mL of saturated NH ₃ / i-PrOH at 55 ° C. for 16 hours. The solvent was then evaporated and the residue was resuspended in a 10% DMSO-containing aqueous solution at pH 7.0 and the HCP carrier was filtered off. The crude product is purified by reverse phase preparative HPLC. Fractions with a purity greater than 95% are collected, concentrated and desalted by reverse phase HPLC. The final desalting product is lyophilized from water.

Table E-34 Outline of oligonucleotide synthesis in DNA / RNA synthesizer ABI-394

ＨＰＬＣ方法５：

Example 84 General purification method:
Buffer A: 50 mM TEAA, pH = 9.6 (adjusted with TEA)
Buffer B: ACN
Column: XBridge Prep C ₁₈ , 5 μm, C ₁₈ , 250 × 10 mm, part number 186003256
Buffer heater set temperature = room temperature Monitor signal at 254 and 280 nm Gradient used:

HPLC method 5:

ONT-60: Oligonucleotide 149 All- (Rp) -d [5mCs8As8Gs8Ts85mCs8Ts8Gs85mCs8Ts8Ts85mCs8G] (s8 is as shown below))

Oligonucleotides were synthesized as described above. RT in RP-HPLC (HPLC method 5): 15.80 minutes. UPLC / ESI-MS: Calculated value: 6345.6; Measured value: 6340.7.

ONT-69: All- (Sp) -d [5mCs16As8Gs8Ts85mCs8Ts8Gs85mCs8Ts8Ts85mCs8G]

Oligonucleotides were synthesized as described above. RT in RP-HPLC (HPLC method 5): 18.61 minutes. UPLC / ESI-MS: Calculated value: 6345.6; Measured value: 6340.6.

Example 85

ABI-394 DNA / RNA synthesis of P-modified blockmer and altmer oligonucleotides containing sterically defined phosphorothioate diesters and sterically defined morpholinoethyl phosphorothioate triester nucleotide linkages. In the apparatus, synthesis was performed using a 1 μmol synthetic column and 1.7 μmol of dG ^{CE, Pac} bound to HCP with oxalyl according to the synthesis cycle summarized in Table E-4. Both sterically defined P-modified phosphorothioate bonds were introduced at predetermined positions in the sequence by performing either the sulfurization step (1) or (2). The synthesis cycle was carried out with the terminal 5'-O-DMTr group removed (DMT Off). The solid support was washed with dehydrated ACN and dried under a stream of argon. The dried HCP was placed in a plastic vial and treated with 1 mL of dehydrated propylamine (ratio 1: 4) in dehydrated pyridine for 18 hours at room temperature. The solvent was then evaporated and the residue was resuspended in DMSO and the HCP carrier was filtered off. The crude product was purified by reverse phase preparative HPLC. Fractions with a purity greater than 95% were collected, concentrated and desalted by reverse phase HPLC (0-80% gradient of ACN). The final desalting product was lyophilized from water.

Table E-4 Outline of oligonucleotide synthesis in the DNA / RNA synthesizer ABI-394 used for the synthesis of Example 85.

Example 86 General Purification Method of Example 85 Buffer A: 20 mM Phosphate pH = 6.0 (adjusted with phosphoric acid)
Buffer B: ACN
Column: XBridge Prep C ₁₈ , 5 μm, C ₁₈ , 250 × 10 mm, product number # 186003256
Buffer solution heater set temperature = 50 ° C
Monitor signals at 254 and 280 nm Gradients used:

HPLC method 6
Buffer A: 20 mM ammonium acetate, pH = 6.0
Buffer B: ACN
Column: XBridge C ₈ , 3.5 μm, 4.6 × 150 mm, product number # 186003034
Buffer solution heater set temperature = 60 ° C
Monitor signals at 254 and 280 nm Gradients used:

ONT-71: All- (Rp) -d [5mCs1As1GsTs5mCsTsGs5mCsTsTs15mCs1G]

Oligonucleotides were synthesized as described above. RT in RP-HPLC (HPLC method 6): 9.39 minutes. UPLC / ESI-MS: Calculated value: 4927.9; Measured value: 4925.3.

ONT-72: All- (Sp) -d [5mCs1As1GsTs5mCsTsGs5mCsTsTs15mCs1G]

Oligonucleotides were synthesized as described above. RT in RP-HPLC (HPLC method 6): 10.84 minutes. UPLC / ESI-MS: Calculated value: 4927.9; Measured value: 4925.7.

ONT-73: All- (Rp) -d [5mCs1AsGs1Ts5mCs1TsGs15mCsTs1Ts5mCs1G]

Oligonucleotides were synthesized as described above. RT in RP-HPLC (HPLC method 6): 13.54 minutes. UPLC / ESI-MS: Calculated value: 4524.2; Measured value: 4522.6.

ONT-74: All- (Sp) -d [5mCs1AsGs1Ts5mCs1TsGs15mCsTs1Ts5mCs1G]

Oligonucleotides were synthesized as described above. RT in RP-HPLC (HPLC method 6): 15.52 minutes. UPLC / ESI-MS: Calculated value: 4524.2; Measured value: 4521.2.

Prodrug oligonucleotide properties such as nuclease resistance, tissue accumulation, cell penetration, endosome escape, immune stimulation, duration of action, pharmacokinetics, etc. are all regulated and influenced by the stereochemistry of chiral phosphorothioate backbone binding.

2'-OH, 2'-OMe, 2'-F, 2'-deoxy, internucleotide phosphodiester or sterically defined internucleotide phosphodiester or sterically defined internucleotide phosphodiester (prodrug) ) (Release either an internucleotide phosphodiester (PO) or a sterically defined internucleotide phosphodiester (PS)) RNA oligonucleotides containing a bond, on an ABI394 DNA / RNA synthesizer, Table E-35. , 5'-O-DMTr-2'-deoxythymidine bound with 130 mg (4.9 μmol) oxalyl prepared as described above according to the cycles summarized in Tables E-36, E-37 and E-38. Synthesize using a packed 10 μmol volume synthetic column. Prior to synthesis, a preliminary capping step (capping 2) is performed to remove the terminal 5'-O-DMTr group to terminate the synthesis. The oxidation step is performed using a commercially available 5-6M decane solution of tert-butyl hydroperoxide (TBHP), which is then diluted with 4 parts of dichloromethane. The stereospecific sulphurization step to the sterically defined internucleotide phosphodiester bond is performed after coupling of the corresponding chiral phosphoramide and a two-step capping step using a 0.3 M methylthiosulfonic acid S-cyanoethyl reagent. Do (Table E-36). The stereospecific sulfide step to a sterically defined internucleotide phosphodiester (PS) bond that releases a sterically defined internucleotide phosphodiester (PS) is 0.3MS- (N-morpholinoethyl thioester-ethyl). )-Para-nitro-toluylthiosulfonate reagent is used after the corresponding chiral phosphoramide coupling and two capping steps (Table E-37). A stereospecific sulphurization step to a sterically defined internucleotide phosphorothioate triester that releases internucleotide phosphodiester (PO) bonds uses a 0.3 M toluylthiosulfonic acid S- (N-morpholinoethyl) reagent. After the corresponding chiral phosphoramide coupling and two capping steps (Table E-38). For 2'-OH RNA nucleotides, 2'-O-PivOM (Debart et al., Chem. Eur. J., 2008, 14, 9135) or 2'-O-TC (Dellinger et al., J. Am. Chem. Use 2'-O-protecting groups such as Soc., 2011, 133, 11540). Ultra-moderate (C-Ac, G-Pac, A-Pac) protecting groups are used for all nucleobases.

表Ｅ－３６ ＤＮＡ／ＲＮＡ合成装置ＡＢＩ３９４におけるオリゴヌクレオチド合成の概要

表Ｅ－３７ ＤＮＡ／ＲＮＡ合成装置ＡＢＩ３９４におけるオリゴヌクレオチド合成の概要

表Ｅ－３８ ＤＮＡ／ＲＮＡ合成装置ＡＢＩ３９４におけるオリゴヌクレオチド合成の概要

Once the automated oligonucleotide synthesis cycle is complete and the final 5'-O-DMTr group has been removed, the synthetic column is removed from the DNA / RNA synthesizer and dried under vacuum. 0.5M 1,5-diazabicyclo (4.3.0) nona-5-ene (DBN) in a 10mL solution, 0.25MN in ACN, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) 1 For minutes, the flow was not stopped and was continuously added to the carrier through the synthetic column using a syringe attached to one end of the synthetic column. The carrier is then washed with anhydrous ACN and dried under vacuum. The dried carrier is then transferred to an empty screw-capped plastic vial and treated with 10% n-PrNH ₂ solution (0.5 mL) in anhydrous pyridine (1.5 mL) for 12 hours at room temperature. The solvent is then evaporated to dryness, the residue containing the solid support is dissolved in 2 mL of water / DMSO (50/50) at pH 5, the carrier is removed by filtration, the backing liquid is recovered and immediately frozen. Store at -80 ° C before purification.

Table E-35 Outline of oligonucleotide synthesis in DNA / RNA synthesizer ABI394

Table E-36 Outline of oligonucleotide synthesis in DNA / RNA synthesizer ABI394

Table E-37 Outline of oligonucleotide synthesis in DNA / RNA synthesizer ABI394

Table E-38 Outline of oligonucleotide synthesis in DNA / RNA synthesizer ABI394

s10 is as follows.

Oligonucleotide Synthesis: (Rp) -uucuAGACcuGuuuuGcuudTs10dT

Synthesis of oligonucleotide ONT-107: (Sp) -uucuAGACcuGuuuuGcuudTs10dT

Synthesis of oligonucleotide ONT-108: (Rp) -AAGcAAAAAcAGGUCuAGAAdTs10dT

Synthesis of oligonucleotide ONT-109: (Sp) -AAGcAAAAAcAGGUCuAGAAdTs10dT

s1 is as follows.

Oligonucleotide Synthesis: (Rp, Rp) -as1AGcAAAAAcAGGUCuAGAAdTsdT

Oligonucleotide Synthesis: (Sp, Rp) -as1GcAAAAAcAGGUCuAGAAdTsdT

Oligonucleotide Synthesis: (Sp, Sp) -as1GcAAAAAcAGGUCuAGAAdTsdT

Oligonucleotide Synthesis: (Rp, Sp) -as1GcAAAAAcAGGUCuAGAAdTsdT

Oligonucleotide Synthesis: (All (Sp))-us1ucus1AGAccs1uGus1uus1uGcuudTs10dT

Oligonucleotide Synthesis: (All (Rp))-As10AGcAAAAAcAGGs1UCuAs1GAs1AdTs10dT

RNA chain thermal annealing and preparation of siRNA double strands. Each RNA strand is mixed with the complementary RNA strand at an equimolar concentration of 10 μM in 1 × PBS. A total 0.5 mL solution is prepared for each double strand, the mixture is heated at 90 ° C. for 2 minutes and allowed to cool for several hours. The mixture is then stored at 4 ° C.

After the thermal RNA strand annealing step, all possible siRNA duplex combinations are prepared by annealing any possible complementary strand of the antisense strand with any sense strand.

After transfection in HeLa or Hep3B cells, all siRNA duplexes prepared are evaluated in vitro for their PCSK9 gene silencing properties.

After free uptake in Hep3B cells, Huh-7 cells or human primary cultured hepatocytes, the prepared siRNA double strands with all prodrug groups are evaluated in vitro for their PCSK9 gene silencing properties.

In combination with a layer of prodrug groups to be further chemically modified and investigated, we observe different efficacy, regulated by the number positions of chiral phosphorothioate backbone bonds and the conformation.

For example, nuclease resistance, cellular penetration, endosome escape, double-stranded thermodynamic stability, double-stranded three-dimensional structure, affinity for various enzymatic interaction mechanism steps, affinity for target mRNAs, specific off-target effects. , Immunostimulation, duration of action, pharmacokinetics, etc. are all regulated and influenced by the stereochemistry of chiral phosphorothioate backbone binding and the presence or absence of prodrug groups.

Addition of PO and PS that release the prodrug group to the siRNA double strand increases its intracellular transport and free uptake in the absence of transfection reagents or target ligands.

Example 87: Stability study of pure oligonucleotides as diastereomers

In this example, the in vitro stability of a chirally pure oligonucleotide and the "original" sterically random mixture (ie, the same sequence as the chirally pure oligonucleotide but not as chirally pure) , For example, compositions containing the resulting oligonucleotides prepared by a sterically random process) are compared for in vitro stability. Pure oligonucleotides are synthesized and dispensed as seven chirals, each with a sequence complementary to the sequence of the gene encoding a particular target transcript or protein of interest, and three different biological matrices (snake venom). Phosphodiesterase (svPDE), nuclease P1 (P1) and rat whole liver homogenate) were used to assess metabolic stability. After different incubation times, levels of full-length oligonucleotides were quantified by IEX-HPLC. The results shown in this example show that, for example, a pure oligonucleotide composition as chiral is a suitable reference (eg, an oligonucleotide of the same sequence but different chiral specificity, particularly comprising a sterically random agent. Drugs), in particular, demonstrate that they can have significantly different metabolic stability compared to the "original" sterically random drug. In this example, oligonucleotides having an antisense sequence on human apolipoprotein-B (ApoB) (thus targeting human apolipoprotein-B (ApoB)) are used to prove the concept in metabolic stability studies. did.

Snake venom phosphodiesterase (svPDE) digestion survey of oligonucleotides ONT-75, ONT-77, ONT-80, ONT-81, ONT-87, ONT-88, ONT-89 and ONT-41

The protocol reported by Oka et al. (J. Am. Chem. Soc. 2008, 130, 16031-16037) was slightly modified and used. Purified oligonucleotides (10 nmol) in water (50 μL) were added to aqueous solution (450 μL, pH 8.6) containing svPDE (4 × 10 ^-3 units), 100 mM Tris HCl and 15 mM MgCl ₂ . The mixture was incubated at 37 ° C. with shaking at 400 rpm. Take 50 μL aliquots at each time point (0h, 12h, 1d, 2d, 3d, 4d, 5d, 6d and 7d), 25 μL 150 mM EDTA, 2 μL Proteinase K (20 mg / mL) and 30 μL lysis buffer. It was deactivated with (Qiagen, # 129115) and the mixture was heated at 60 ° C. for 20 minutes. 5 μL of internal standard (5' -GC GTT TGCTCTT CTTCTTGCGTTTT TTT -3', 27-mer oligonucleotides (underlined nucleotides are 2'-MOE modified), (200 μM) were added to the above aliquots. Quantitative analysis IEX. -HPLC was performed and metabolite identification was performed by UPLC / MS. The results are shown in FIG. 59.

IEX-HPLC analysis showed degradation of the phosphorothioate dimer during incubation with svPDE and showed no significant difference between the stereoisomers. LCMS analysis of metabolites revealed that the majority of degradation products were formed as a result of desulfurization. 2'-MOE at the 5'and 3'-ends, as previously reported by Prakash et al. (Biochemistry 2002, 41, 11642-11648), Prhavc et al. (Org. Lett., 2003, 5, 2017-2020) and others. We also observed that the modification protects these oligomers from svPDE metabolism, which is an exonuclease in the 3'to 5'direction.

Nuclease P1 (P1) digestion study of oligonucleotides ONT-75, ONT-77, ONT-80, ONT-81, ONT-87, ONT-88, ONT-89 and ONT-41

The protocol reported by Oka et al. (J. Am. Chem. Soc. 2008, 130, 16031-16037) was slightly modified and used. Purified oligonucleotides (10 nmol) in water (50 μL) were added to an aqueous solution (500 μL, pH 7.2) containing nP1 (20 units), 100 mM Tris HCl and 1 mM ZnCl ₂ . The mixture was incubated at 37 ° C. with shaking at 400 rpm. Take 50 μL aliquots at each time point (0h, 1h, 2h, 4h, 8h, 12h, 1d, 2d) and take 25 μL stop buffer (150 mM EDTA, 2 μL Proteinase K solution (20 mg / mL) and 30 μL. It was deactivated with lysis buffer (Qiagen, # 129115)) and the mixture was heated at 60 ° C. for 20 minutes. 5 μL of internal standard (5'- GC GTTT GCTTT CTTCTTGCGTTTT TT -3'), 27-mer oligonucleotides (underlined nucleotides are 2'-MOE modified) (200 μM) were added to the above aliquots. Quantitative analysis was performed by IEX-HPLC and metabolite identification was performed by UPLC / MS. The results are shown in FIGS. 60-67.

It has been previously reported that nuclease _nP1 specifically cleaves a DNA phosphorothioate bond with an absolute configuration (SP) at the phosphorus atom (Porter et al., Biochemistry, 1983, 22, 1369-1377; Oka et al., J. . Am. Chem. Soc., 2008, 130, 16031-16037). Similar cleavage patterns were observed with the 5-10-5 2'-MOE gapmer phosphorothioate oligonucleotides investigated and the gapmer oligonucleotides at the (SP) phosphorothioate center without _nP1 affecting the 2'-MOE wings. It was revealed that the DNA nuclei of the 2'-MOE wings were efficiently digested, and the 2'-MOE wings were stable without depending on stereochemistry. A sterically random diastereomix oligonucleotide ONT-41, as well as sterically pure oligonucleotides containing (SP) -phosphorothioate nucleotide linkages in the DNA nucleus (ONT _- 77, ONT-80, ONT-87, Complete digestion of DNA nuclei within 1 hour was observed with ONT-88 and ONT-89). The total product of cleavage was clearly identified by UPLC / MS. As previously reported in the literature, it has been found that apparently ( _RP ) phosphorothioate DNA is not a substrate for nP1. Two chirally pure oligonucleotides (ONT-75 and ONT-81) with ( _RP ) phosphorothioate DNA nuclei were completely stable to nP1 during 1 hour incubation at 37 ° C. ONT-75 and ONT-81 showed a loss of about 10-15% full length product over several days of incubation. The results shown in this example show that, for example, a pure oligonucleotide composition as chiral is a suitable reference (eg, an oligonucleotide of the same sequence but different chiral specificity, particularly comprising a sterically random agent. Drugs), specifically compared to "original" sterically random drugs, demonstrate that they can have significantly different metabolic stability in the nP1 assay.

General IEX-HPLC Method for Analysis of Enzyme Digestion Products Buffer A: 10 mM Tris HCl, 50% ACN, pH 8.0
Buffer B: 10 mM Tris HCl, 800 mM NaClO ₄ , 50% ACN, pH = 8.0
Column: DIONEX, DNAPac, PA-100, 4.0 x 250 mm, product number # 063000
Column temperature = 60 ° C
Monitor signals at 254 and 280 nm Gradients used:

Common UPLC-LCMS Methods for Analysis of Enzyme Digestion Products Buffer A: 15 mM TEA, 400 mM HFIP, Water Buffer B: 50:50 Buffer A / Methanol Column: UPLC @ OST C ₁₈ 1.7 μm, 2 .1 x 500 mm
Column temperature = 60 ° C
Gradient used:

In vitro metabolic stability of pure human oligonucleotides as diastereomers in pre-incubated rat whole liver homogenates

The metabolic stability of chirally pure oligonucleotides in in vitro rat whole liver homogenates was measured. The protocol used here has been previously reported and has been used to assess the stability of oligonucleotide agents.

Protocol: In this study, the protocol reported by Geary et al. Was slightly modified and used (Oligonucleotides, 2010, 20, 309).

Test system: Two male Sprague-Dawley rats (Rattus norvegicus) were supplied by Charles River Laboratories, Inc. (Hollister, CA, CA).

Tissue harvesting: Animals were acclimatized to the laboratory for 2 days prior to tissue harvesting. At the time of tissue collection, animals were anesthetized by intraperitoneal (IP) injection of pentobarbital sodium solution. Liver perfusion was performed using 500 mL of chilled saturated saline / animal administered from the hepatic portal vein. After perfusion, the liver was dissected and maintained on ice. The liver was chopped into small pieces and weighed.

Liver homogenate preparation: Chopped pieces of liver tissue were transferred to a tare weighed 50 mL centrifuge tube and weighed. Cooled homogenizing buffer (100 mL Tris pH 8.0, 1 mM magnesium acetate, and antibacterial-antifungal agent) was added to each tube so that the tube contained 5 mL of buffer per 1 g of tissue. The QIAGEN TissueRuptor tissue homogenizer was used to homogenize the liver / buffer mixture, while the tubes were maintained on ice. The protein concentration of liver homogenate was determined using the Pierce BCA protein assay. Liver homogenate was divided into 1 mL aliquots, transferred to Cryovial and stored at -60 ° C.

Incubation conditions: A fixed amount of 1 mL of frozen liver homogenate (protein concentration = 31.9 mg / mL) was thawed and incubated at 37 ° C. for 24 hours. Six Eppendorf tubes (2 mL) were taken and 450 μL of homogenate was added to each tube. 50 μL of test oligonucleotide (200 μL) was added to each tube. Immediately after mixing, 125 μL (5 ×) stop buffer (2.5% IGEPAL, 0.5M NaCl, 5 mM EDTA, 50 mM Tris, pH = 8.0) and 12.5 μL 20 mg / mL proteinase K (Ambion,). # 2546) was added to one tube as a time point of 0 h. The mixture was then heated at 60 ° C. for 1 hour. The remaining reaction mixture was incubated at 37 ° C. with a VWR incubating microplate shaker, shaking at 400 rpm. After incubation for a predetermined period (1, 2, 3, 4 and 5 days), 125 μL (5 ×) stop buffer (2.5% IGEPA, 0.5M NaCl, 5 mM EDTA, 50 mM Tris, pH) of each mixture. = 8.0) and 12.5 μL of 20 mg / mL proteinase K (Ambion, # 2546).

Post-treatment and bioanalysis: (5'- GC GTTT GCTTT CTTCTTGCGTTTT TT -3'), 27-mer oligonucleotides (underlined nucleotides are 2'-MOE-modified) for quantification of pure oligonucleotides as diastereomers. Used as an internal standard. 50 μL of internal standard (200 μM) was added to each tube, followed by 250 μL of 30% ammonium hydroxide, 800 μL of phenol: chloroform: isoamyl alcohol (25:24: 1). After mixing and centrifuging at 600 rpm, the aqueous layer was evaporated with SpeedVac to 100 μL and packed in a SepPak column (C18, 1 g, WAT036905). All aqueous lotions on the Sep Pak column were tested by rapid IEX-HPLC and confirmed to be no product. The acetonitrile eluate was concentrated, dried, dissolved in 100 μL of water and analyzed using RP-HPLC.
Eluent A: 10 mM tributylammonium acetate, pH = 7.0
Eluent B: ACN
Column: XTerra MS C ₁₈ , 3.5 μm, 4.6 × 150 mm, Part No. 186000432
Column temperature = 60 ° C
HPLC gradient:

FIG. 68 shows that different chirally pure oligonucleotides have different metabolic stability profiles in rat whole liver homogenates. Experimentally, purely sterically defined oligonucleotides (here, ONT-75 and ONT-77 are used here as examples) as diastereomers with the same sequence and chemical composition, but as understood by those skilled in the art. , This observation can be inferred from the purely sterically defined phosphorothioate diastereoisomers as other possible diastereomers of this molecule), the diastereoisomer mixture ONT-41 has a different metabolic stability. It also demonstrates having a sex profile.

Oligonucleotide ONT-75 with a sterically controlled phosphorothioate backbone with a total _RP absolute configuration in all phosphorus atoms was completely degraded in one day in rat whole liver homogenates preincubated at 37 ° C. and used in this study. It showed the lowest metabolic stability of the three.

The phosphorothioate oligonucleotide ONT-77 (with absolute configuration: 5R-10S-4R), which is purely sterically defined as another diastereomer, is compared to the sterically random ONT-41 (mipomersen). It was more stable than double. After 5 days of incubation, 40% full-length oligonucleotides remained in pure ONT-77 as diastereomers, whereas about 15% full-length oligonucleotides remained in sterically random ONT-41 (mipomersen). .. A direct comparison of ONT-77 and ONT-41 revealed that the sterically controlled isomer 5R-10S-4R (ONT-77) clearly showed significantly higher metabolic stability throughout all analytical time points. The sterically defined structure of the pure isomer ONT-77 as a diastereomer affects the rate of degradation and overall metabolic stability of this oligonucleotide. Although not intended to be bound by theory, SP stereochemistry applied to the DNA nuclei of 5-10-5 _gapmer oligonucleotides is endonuclease compared to sterically random diastereomixes. These observations lead to the conclusion that they provide increased resistance and thus increase metabolic stability. It would be expected that some other conformation of this sequence, as other diastereomers pure stereoregulated phosphorothioate isomers, would exhibit even higher metabolic stability in this experiment.

Although not intended to be constrained by theory, the results shown in this example show that, for example, _RP stereochemistry with phosphorothioate binding (ONT _- 75) contains SP DNA nuclei in rat liver homogenates. Demonstrate that the metabolic stability is lower than that of ONT-77. The results shown in this example show that, for example, a pure oligonucleotide composition as chiral is a suitable reference (eg, an oligonucleotide of the same sequence but different chiral specificity, particularly comprising a sterically random agent. It also demonstrates that it may have significantly different metabolic stability in rat liver homogenates as compared to the drug), in particular as compared to the "original" sterically random drug. In this example, in this example, an oligonucleotide having an antisense sequence on human apolipoprotein-B (ApoB) (hence targeting human apolipoprotein-B (ApoB)) is metabolically stable. Used for proof of concept in the survey. The chiral-controlled oligonucleosides provided may have increased or decreased stability to the endogenous enzyme. By providing chiral-controlled oligonucleotides and compositions and methods thereof, the present invention provides known non-chiral-controlled oligonucleotides and oligonucleotides and compositions with increased pharmacological properties over their compositions. Provided.

In vitro metabolic stability of human PCSK9 siRNA double strand with sterically controlled phosphodiester bond in human serum

Use a protocol similar to the previously reported procedure (Oka et al., J. Am. Chem. Soc. 2008, 130, 16031-16037). SiRNA double strand (2.5 μmol) in water (50 μL) is added to human serum (450 μL). Incubate the mixture at 37 ° C. with shaking at 400 rpm. Take 50 μL aliquots at each time point (0h, 0.5h, 1h, 2h, 4h, 6h, 8h and 24h), 25 μL 150 mM EDTA, 2 μL Proteinase K solution (20 mg / mL) and 30 μL lysis buffer. Inactivate with liquid (Qiagen, # 129115) and heat the mixture at 60 ° C. for 20 minutes. Add 5 μL of an internal standard (5' -GC GTT TGCTCTT CTTCTTGCGTTTT TTT -3', 27-mer oligonucleotide (underlined nucleotides are 2'-MOE modified)) (200 μM) to the above aliquot. Quantitative analysis is performed by IEX-HPLC and metabolite identification is performed by UPLC / MS.

In some embodiments, siRNA double strands with pure phosphorothioates as 3'end diastereomers with _SP arrangements are phosphorothioates with _RP arrangements at the same position or sterically random diastereomixtures. It shows high metabolic stability in human serum as compared with siRNA having. In other embodiments, the siRNA double strand having a pure phosphorothioate as a 5'end diastereomer with an _SP configuration has a phosphorothioate with an _RP configuration at the same position or a sterically random diastereomixture. Shows higher metabolic stability in human serum compared to siRNA. In other embodiments, siRNA double strands with pure phosphorothioates as diastereomers with SP configurations at both the 3'and _5'ends are phosphorothioates with _RP configurations at the same position or sterically random. It exhibits high metabolic stability in human serum compared to siRNA with a diastereomix. In other embodiments, siRNA double strands with pure phosphorothioates as multiple diastereomers with _SP arrangements are compared to siRNAs with multiple phosphorothioates with _RP arrangements or sterically random diastereomixes. As a result, it exhibits high metabolic stability in human serum. In other embodiments, the siRNA double strand having the entire backbone of the pure phosphorothioate as a diastereomer with the _SP configuration is the siRNA having the entire backbone of the phosphorothioate having the _RP configuration or a sterically random diastereomixture. Shows higher metabolic stability in human serum.

Example 88 Chiral-controlled oligonucleotide compositions show different in vitro efficacy as compared to non-chirally controlled compositions having the same sequence.

In this example, the in vitro pharmacological activity of a chirally pure oligonucleotide is expressed in an "original" sterically random mixture (ie, the same sequence as the chirally pure oligonucleotide, but not as chirally pure. No, eg, a composition containing the resulting oligonucleotide prepared by a sterically random process) compared to the activity observed. Pure oligonucleotides as four chirals, each with a sequence complementary to the sequence of the gene encoding a particular target transcript or protein of interest, were synthesized, dispensed and transfected into primary mouse hepatocytes. The mRNA level was quantified and the suppression level was evaluated. In this example, an oligonucleotide having an antisense sequence on human apolipoprotein-B (ApoB) (hence targeting human apolipoprotein-B (ApoB)) is used to demonstrate the concept of transgenic mouse-expressing human ApoB. Used for.

Transfection of mouse primary hepatocytes with oligonucleotides Using 7-week-old C57BL6 male mice, mouse primary hepatocytes were extracted and sown on 96-well plates (no overlay) (Celsis / Bioreclamation IV). .. Transfection of primary hepatocytes was performed using lipofectin (Life Technologies, stock number 18292-037) and 0.5 μl lipofectin per 96-plate well using the manufacturer's protocol. Starting from 6 μM, 12 1: 3 siRNA double-stranded diluents were prepared. 10 μl of 10 × oligo was then lipoplexed with 9.5 μl of in vitro Gro HI Medium (Celsis, stock number Z99009) serum-free medium and 0.5 μl of lipofectin preparation mixture per well. After 10-15 minutes of incubation, 20 μl of lipoplexed oligo was added to 80 μl of in vitro Gro HI Medium primary hepatocytes to a final volume of 100 μl per well. Twenty-four hours after transfection, cells were lysed.

Apolypoprotein B mRNA assay Using MagMAX ™ -96 Total RNA Isolation Kit (Life Technologies, AM1830), total mRNA was purified from cell solubilized products; high volume cDNA reverse transcription kit with RNase inhibitor ( Life Technologies (4374967) synthesized 15 μl of cDNA. Gene expression was evaluated by real-time PCR on Lightcycller 480 (Roche) using the following primers using the Probes Master Mix (Roche, 04 707 494 001) according to the manufacturer's protocol.
Mouse Apolipoprotein B Primer:
[Template: (GenBank catalog number M35186)]
-Forward primer: CGTGGGCTCCAGCATTCTA
-Reverse primer: AGTCATTCTGCCCTTGCGTC
-PCR probe: FAM-CCAATGGTCGGGCACTGCTCAA-TAMRA
Mouse GAPDH Primer-Forward Primer: GGCAAATTCAAGGCACAGT
-Reverse primer: GGGTTCCGCTCCTGGAAGAT
-PCR probe: 5'JOE-AAGGCCGAGAAGGAAGCTTGTTCATC-TAMRA3'

Data analysis The values were calculated using the Delta Delta Ct method. For each sample, the mean ApoB signal was normalized to the mean GAPDH signal and then normalized to the corresponding mean ratios of sham-transfected and untreated samples to give relative levels of ApoB protein expression. An "original" sterically random mixture was used as a reference. To calculate relative IC50s using Graphpad prisms, a four-parameter linear regression curve was fitted to the data and the bottom and top were constrained to constants 0 and 100, respectively.

In vitro dosing responses (FIGS. 71 and 72) show that pure oligonucleotides as chiral have significantly different efficacy, with ONT-83 being the most effective and ONT-84 and ONT-85 being the most effective. It is less sexual and shows a 8.6-fold difference in IC50 (Table E-39).

Table E-39 IC50 values of stereoisomers (ONT-83, -84, -85 or -86) for suppression of mouse apolipoprotein B / GAPDH mRNA levels in primary mouse hepatocytes

The results shown in this example show that, for example, an oligonucleotide composition pure as chiral is a suitable reference (eg, an oligonucleotide of the same sequence but different chiral specificity, particularly comprising a sterically random agent. Drugs), in particular, demonstrate that they can have significantly different pharmacological activities in vitro compared to "original" sterically random drugs. Those skilled in the art will appreciate that the chiral controlled oligonucleotide compositions provided by the present disclosure have unexpected activity and properties in view of this demonstration.

Doctrine of equivalents

Although some exemplary embodiments of the invention have been described, those skilled in the art will appreciate that the above are merely exemplary, not limiting, and are represented only by way of example. Will be obvious. Many modifications and other exemplary embodiments are within the scope of those skilled in the art and are intended to be within the scope of the present invention. In particular, many embodiments presented herein include specific combinations or system elements of method actions, but these actions and these elements are combined in other ways to achieve the same purpose. It should be understood that it can be done. Actions, elements, and features discussed only with respect to one embodiment are not intended to be excluded from similar roles in other embodiments. In addition, because of one or more means + function limitations cited in the following claims, the means are disclosed herein to perform the cited functions. It is not intended to limit the means of the above, but to include any means now known or later developed for the performance of the citation function.

The use of ordinary words in the claims, such as "first," "second," "third," and others, to modify the elements of the claims, in and of itself, has any priority. , Or an order of one claim element to another, or that temporal order of that act of the method is enforced, but one claim element with a particular name. Is used only as a marker to identify an element in the claims and to another element having the same name (but for the use of ordinary terms). Similarly, a), b), others, or i), ii), other uses themselves do not imply any priorities, priorities, or step orders within the scope of this claim. Similarly, the use of these terms herein does not imply any required priority, precedence, or order in and of itself.

添付資料 (B)

添付資料 (C)

代表的なヒトmiRNA配列

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MI0000234GCCGAGACCGAGUGCACAGGGCUCUGACCUAUGAAUUGACAGCCAGUGCUCUCGUCUCCCCUCUGGCUGCCAAUUCCAUAGGUCACAGGUAUGUUCGCCUCAAUGCCAGC>hsa-mir-193a MI0000487CGAGGAUGGGAGCUGAGGGCUGGGUCUUUGCGGGCGAGAUGAGGGUGUCGGAUCAACUGGCCUACAAAGUCCCAGUUCUCGGCCCCCG>hsa-mir-193b MI0003137GUGGUCUCAGAAUCGGGGUUUUGAGGGCGAGAUGAGUUUAUGUUUUAUCCAACUGGCCCUCAAAGUCCCGCUUUUGGGGUCAU>hsa-mir-194-1 MI0000488AUGGUGUUAUCAAGUGUAACAGCAACUCCAUGUGGACUGUGUACCAAUUUCCAGUGGAGAUGCUGUUACUUUUGAUGGUUACCAA>hsa-mir-194-2 MI0000732UGGUUCCCGCCCCCUGUAACAGCAACUCCAUGUGGAAGUGCCCACUGGUUCCAGUGGGGCUGCUGUUAUCUGGGGCGAGGGCCAG>hsa-mir-195 MI0000489AGCUUCCCUGGCUCUAGCAGCACAGAAAUAUUGGCACAGGGAAGCGAGUCUGCCAAUAUUGGCUGUGCUGCUCCAGGCAGGGUGGUG>hsa-mir-196a-1 MI0000238GUGAAUUAGGUAGUUUCAUGUUGUUGGGCCUGGGUUUCUGAACACAACAACAUUAAACCACCCGAUUCAC>hsa-mir-196a-2 MI0000279UGCUCGCUCAGCUGAUCUGUGGCUUAGGUAGUUUCAUGUUGUUGGGAUUGAGUUUUGAACUCGGCAACAAGAAACUGCCUGAGUUACAUCAGUCGGUUUUCGUCGAGGGC>hsa-mir-196b MI0001150ACUGGUCGGUGAUUUAGGUAGUUUCCUGUUGUUGGGAUCCACCUUUCUCUCGACAGCACGACACUGCCUUCAUUACUUCAGUUG>hsa-mir-197 MI0000239GGCUGUGCCGGGUAGAGAGGGCAGUGGGAGGUAAGAGCUCUUCACCCUUCACCACCUUCUCCACCCAGCAUGGCC>hsa-mir-198 MI0000240UCAUUGGUCCAGAGGGGAGAUAGGUUCCUGUGAUUUUUCCUUCUUCUCUAUAGAAUAAAUGA>hsa-mir-199a-1 MI0000242GCCAACCCAGUGUUCAGACUACCUGUUCAGGAGGCUCUCAAUGUGUACAGUAGUCUGCACAUUGGUUAGGC>hsa-mir-199a-2 MI0000281AGGAAGCUUCUGGAGAUCCUGCUCCGUCGCCCCAGUGUUCAGACUACCUGUUCAGGACAAUGCCGUUGUACAGUAGUCUGCACAUUGGUUAGACUGGGCAAGGGAGAGCA>hsa-mir-199b MI0000282CCAGAGGACACCUCCACUCCGUCUACCCAGUGUUUAGACUAUCUGUUCAGGACUCCCAAAUUGUACAGUAGUCUGCACAUUGGUUAGGCUGGGCUGGGUUAGACCCUCGG>hsa-mir-200a MI0000737CCGGGCCCCUGUGAGCAUCUUACCGGACAGUGCUGGAUUUCCCAGCUUGACUCUAACACUGUCUGGUAACGAUGUUCAAAGGUGACCCGC>hsa-mir-200b MI0000342CCAGCUCGGGCAGCCGUGGCCAUCUUACUGGGCAGCAUUGGAUGGAGUCAGGUCUCUAAUACUGCCUGGUAAUGAUGACGGCGGAGCCCUGCACG>hsa-mir-200c MI0000650CCCUCGUCUUACCCAGCAGUGUUUGGGUGCGGUUGGGAGUCUCUAAUACUGCCGGGUAAUGAUGGAGG>hsa-mir-202 MI0003130CGCCUCAGAGCCGCCCGCCGUUCCUUUUUCCUAUGCAUAUACUUCUUUGAGGAUCUGGCCUAAAGAGGUAUAGGGCAUGGGAAAACGGGGCGGUCGGGUCCUCCCCAGCG>hsa-mir-203 MI0000283GUGUUGGGGACUCGCGCGCUGGGUCCAGUGGUUCUUAACAGUUCAACAGUUCUGUAGCGCAAUUGUGAAAUGUUUAGGACCACUAGACCCGGCGGGCGCGGCGACAGCGA>hsa-mir-204 MI0000284GGCUACAGUCUUUCUUCAUGUGACUCGUGGACUUCCCUUUGUCAUCCUAUGCCUGAGAAUAUAUGAAGGAGGCUGGGAAGGCAAAGGGACGUUCAAUUGUCAUCACUGGC>hsa-mir-205 MI0000285AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAUGGAGCUGACA>hsa-mir-206 MI0000490UGCUUCCCGAGGCCACAUGCUUCUUUAUAUCCCCAUAUGGAUUACUUUGCUAUGGAAUGUAAGGAAGUGUGUGGUUUCGGCAAGUG>hsa-mir-208a MI0000251UGACGGGCGAGCUUUUGGCCCGGGUUAUACCUGAUGCUCACGUAUAAGACGAGCAAAAAGCUUGUUGGUCA>hsa-mir-208b MI0005570CCUCUCAGGGAAGCUUUUUGCUCGAAUUAUGUUUCUGAUCCGAAUAUAAGACGAACAAAAGGUUUGUCUGAGGGCAG>hsa-mir-210 MI0000286ACCCGGCAGUGCCUCCAGGCGCAGGGCAGCCCCUGCCCACCGCACACUGCGCUGCCCCAGACCCACUGUGCGUGUGACAGCGGCUGAUCUGUGCCUGGGCAGCGCGACCC>hsa-mir-211 MI0000287UCACCUGGCCAUGUGACUUGUGGGCUUCCCUUUGUCAUCCUUCGCCUAGGGCUCUGAGCAGGGCAGGGACAGCAAAGGGGUGCUCAGUUGUCACUUCCCACAGCACGGAG>hsa-mir-212 MI0000288CGGGGCACCCCGCCCGGACAGCGCGCCGGCACCUUGGCUCUAGACUGCUUACUGCCCGGGCCGCCCUCAGUAACAGUCUCCAGUCACGGCCACCGACGCCUGGCCCCGCC>hsa-mir-214 MI0000290GGCCUGGCUGGACAGAGUUGUCAUGUGUCUGCCUGUCUACACUUGCUGUGCAGAACAUCCGCUCACCUGUACAGCAGGCACAGACAGGCAGUCACAUGACAACCCAGCCU>hsa-mir-215 MI0000291AUCAUUCAGAAAUGGUAUACAGGAAAAUGACCUAUGAAUUGACAGACAAUAUAGCUGAGUUUGUCUGUCAUUUCUUUAGGCCAAUAUUCUGUAUGACUGUGCUACUUCAA>hsa-mir-216a MI0000292GAUGGCUGUGAGUUGGCUUAAUCUCAGCUGGCAACUGUGAGAUGUUCAUACAAUCCCUCACAGUGGUCUCUGGGAUUAUGCUAAACAGAGCAAUUUCCUAGCCCUCACGA>hsa-mir-216b MI0005569GCAGACUGGAAAAUCUCUGCAGGCAAAU
GUGAUGUCACUGAGGAAAUCACACACUUACCCGUAGAGAUUCUACAGUCUGACA>hsa-mir-217 MI0000293AGUAUAAUUAUUACAUAGUUUUUGAUGUCGCAGAUACUGCAUCAGGAACUGAUUGGAUAAGAAUCAGUCACCAUCAGUUCCUAAUGCAUUGCCUUCAGCAUCUAAACAAG>hsa-mir-218-1 MI0000294GUGAUAAUGUAGCGAGAUUUUCUGUUGUGCUUGAUCUAACCAUGUGGUUGCGAGGUAUGAGUAAAACAUGGUUCCGUCAAGCACCAUGGAACGUCACGCAGCUUUCUACA>hsa-mir-218-2 MI0000295GACCAGUCGCUGCGGGGCUUUCCUUUGUGCUUGAUCUAACCAUGUGGUGGAACGAUGGAAACGGAACAUGGUUCUGUCAAGCACCGCGGAAAGCACCGUGCUCUCCUGCA>hsa-mir-219-1 MI0000296CCGCCCCGGGCCGCGGCUCCUGAUUGUCCAAACGCAAUUCUCGAGUCUAUGGCUCCGGCCGAGAGUUGAGUCUGGACGUCCCGAGCCGCCGCCCCCAAACCUCGAGCGGG>hsa-mir-219-2 MI0000740ACUCAGGGGCUUCGCCACUGAUUGUCCAAACGCAAUUCUUGUACGAGUCUGCGGCCAACCGAGAAUUGUGGCUGGACAUCUGUGGCUGAGCUCCGGG>hsa-mir-221 MI0000298UGAACAUCCAGGUCUGGGGCAUGAACCUGGCAUACAAUGUAGAUUUCUGUGUUCGUUAGGCAACAGCUACAUUGUCUGCUGGGUUUCAGGCUACCUGGAAACAUGUUCUC>hsa-mir-222 MI0000299GCUGCUGGAAGGUGUAGGUACCCUCAAUGGCUCAGUAGCCAGUGUAGAUCCUGUCUUUCGUAAUCAGCAGCUACAUCUGGCUACUGGGUCUCUGAUGGCAUCUUCUAGCU>hsa-mir-223 MI0000300CCUGGCCUCCUGCAGUGCCACGCUCCGUGUAUUUGACAAGCUGAGUUGGACACUCCAUGUGGUAGAGUGUCAGUUUGUCAAAUACCCCAAGUGCGGCACAUGCUUACCAG>hsa-mir-224 MI0000301GGGCUUUCAAGUCACUAGUGGUUCCGUUUAGUAGAUGAUUGUGCAUUGUUUCAAAAUGGUGCCCUAGUGACUACAAAGCCC>hsa-mir-296 MI0000747AGGACCCUUCCAGAGGGCCCCCCCUCAAUCCUGUUGUGCCUAAUUCAGAGGGUUGGGUGGAGGCUCUCCUGAAGGGCUCU>hsa-mir-297 MI0005775UGUAUGUAUGUGUGCAUGUGCAUGUAUGUGUAUAUACAUAUAUAUGUAUUAUGUACUCAUAUAUCA>hsa-mir-298 MI0005523UCAGGUCUUCAGCAGAAGCAGGGAGGUUCUCCCAGUGGUUUUCCUUGACUGUGAGGAACUAGCCUGCUGCUUUGCUCAGGAGUGAGCU>hsa-mir-299 MI0000744AAGAAAUGGUUUACCGUCCCACAUACAUUUUGAAUAUGUAUGUGGGAUGGUAAACCGCUUCUU>hsa-mir-300 MI0005525UGCUACUUGAAGAGAGGUAAUCCUUCACGCAUUUGCUUUACUUGCAAUGAUUAUACAAGGGCAGACUCUCUCUGGGGAGCAAA>hsa-mir-301a MI0000745ACUGCUAACGAAUGCUCUGACUUUAUUGCACUACUGUACUUUACAGCUAGCAGUGCAAUAGUAUUGUCAAAGCAUCUGAAAGCAGG>hsa-mir-301b MI0005568GCCGCAGGUGCUCUGACGAGGUUGCACUACUGUGCUCUGAGAAGCAGUGCAAUGAUAUUGUCAAAGCAUCUGGGACCA>hsa-mir-302a 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MI0003778UUUGCAUUAAAAAUGAGGCCUUCUCUUCCCAGUUCUUCCCAGAGUCAGGAAAAGCUGGGUUGAGAGGGUAGAAAAAAAAUGAUGUAGG>hsa-mir-320c-2 MI0008191CUUCUCUUUCCAGUUCUUCCCAGAAUUGGGAAAAGCUGGGUUGAGAGGGU>hsa-mir-320d-1 MI0008190UUCUCGUCCCAGUUCUUCCCAAAGUUGAGAAAAGCUGGGUUGAGAGGA>hsa-mir-320d-2 MI0008192UUCUCUUCCCAGUUCUUCUUGGAGUCAGGAAAAGCUGGGUUGAGAGGA>hsa-mir-320e MI0014234GCCUUCUCUUCCCAGUUCUUCCUGGAGUCGGGGAAAAGCUGGGUUGAGAAGGU>hsa-mir-323a MI0000807UUGGUACUUGGAGAGAGGUGGUCCGUGGCGCGUUCGCUUUAUUUAUGGCGCACAUUACACGGUCGACCUCUUUGCAGUAUCUAAUC>hsa-mir-323b MI0014206UGGUACUCGGAGGGAGGUUGUCCGUGGUGAGUUCGCAUUAUUUAAUGAUGCCCAAUACACGGUCGACCUCUUUUCGGUAUCA>hsa-mir-324 MI0000813CUGACUAUGCCUCCCCGCAUCCCCUAGGGCAUUGGUGUAAAGCUGGAGACCCACUGCCCCAGGUGCUGCUGGGGGUUGUAGUC>hsa-mir-325 MI0000824AUACAGUGCUUGGUUCCUAGUAGGUGUCCAGUAAGUGUUUGUGACAUAAUUUGUUUAUUGAGGACCUCCUAUCAAUCAAGCACUGUGCUAGGCUCUGG>hsa-mir-326 MI0000808CUCAUCUGUCUGUUGGGCUGGAGGCAGGGCCUUUGUGAAGGCGGGUGGUGCUCAGAUCGCCUCUGGGCCCUUCCUCCAGCCCCGAGGCGGAUUCA>hsa-mir-328 MI0000804UGGAGUGGGGGGGCAGGAGGGGCUCAGGGAGAAAGUGCAUACAGCCCCUGGCCCUCUCUGCCCUUCCGUCCCCUG>hsa-mir-329-1 MI0001725GGUACCUGAAGAGAGGUUUUCUGGGUUUCUGUUUCUUUAAUGAGGACGAAACACACCUGGUUAACCUCUUUUCCAGUAUC>hsa-mir-329-2 MI0001726GUGGUACCUGAAGAGAGGUUUUCUGGGUUUCUGUUUCUUUAUUGAGGACGAAACACACCUGGUUAACCUCUUUUCCAGUAUCAA>hsa-mir-330 MI0000803CUUUGGCGAUCACUGCCUCUCUGGGCCUGUGUCUUAGGCUCUGCAAGAUCAACCGAGCAAAGCACACGGCCUGCAGAGAGGCAGCGCUCUGCCC>hsa-mir-331 MI0000812GAGUUUGGUUUUGUUUGGGUUUGUUCUAGGUAUGGUCCCAGGGAUCCCAGAUCAAACCAGGCCCCUGGGCCUAUCCUAGAACCAACCUAAGCUC>hsa-mir-335 MI0000816UGUUUUGAGCGGGGGUCAAGAGCAAUAACGAAAAAUGUUUGUCAUAAACCGUUUUUCAUUAUUGCUCCUGACCUCCUCUCAUUUGCUAUAUUCA>hsa-mir-337 MI0000806GUAGUCAGUAGUUGGGGGGUGGGAACGGCUUCAUACAGGAGUUGAUGCACAGUUAUCCAGCUCCUAUAUGAUGCCUUUCUUCAUCCCCUUCAA>hsa-mir-338 MI0000814UCUCCAACAAUAUCCUGGUGCUGAGUGAUGACUCAGGCGACUCCAGCAUCAGUGAUUUUGUUGAAGA>hsa-mir-339 MI0000815CGGGGCGGCCGCUCUCCCUGUCCUCCAGGAGCUCACGUGUGCCUGCCUGUGAGCGCCUCGACGACAGAGCCGGCGCCUGCCCCAGUGUCUGCGC>hsa-mir-340 MI0000802UUGUACCUGGUGUGAUUAUAAAGCAAUGAGACUGAUUGUCAUAUGUCGUUUGUGGGAUCCGUCUCAGUUACUUUAUAGCCAUACCUGGUAUCUUA>hsa-mir-342 MI0000805GAAACUGGGCUCAAGGUGAGGGGUGCUAUCUGUGAUUGAGGGACAUGGUUAAUGGAAUUGUCUCACACAGAAAUCGCACCCGUCACCUUGGCCUACUUA>hsa-mir-345 MI0000825ACCCAAACCCUAGGUCUGCUGACUCCUAGUCCAGGGCUCGUGAUGGCUGGUGGGCCCUGAACGAGGGGUCUGGAGGCCUGGGUUUGAAUAUCGACAGC>hsa-mir-346 MI0000826GGUCUCUGUGUUGGGCGUCUGUCUGCCCGCAUGCCUGCCUCUCUGUUGCUCUGAAGGAGGCAGGGGCUGGGCCUGCAGCUGCCUGGGCAGAGCGG>hsa-mir-361 MI0000760GGAGCUUAUCAGAAUCUCCAGGGGUACUUUAUAAUUUCAAAAAGUCCCCCAGGUGUGAUUCUGAUUUGCUUC>hsa-mir-362 MI0000762CUUGAAUCCUUGGAACCUAGGUGUGAGUGCUAUUUCAGUGCAACACACCUAUUCAAGGAUUCAAA>hsa-mir-363 MI0000764UGUUGUCGGGUGGAUCACGAUGCAAUUUUGAUGAGUAUCAUAGGAGAAAAAUUGCACGGUAUCCAUCUGUAAACC>hsa-mir-365a MI0000767ACCGCAGGGAAAAUGAGGGACUUUUGGGGGCAGAUGUGUUUCCAUUCCACUAUCAUAAUGCCCCUAAAAAUCCUUAUUGCUCUUGCA>hsa-mir-365b MI0000769AGAGUGUUCAAGGACAGCAAGAAAAAUGAGGGACUUUCAGGGGCAGCUGUGUUUUCUGACUCAGUCAUAAUGCCCCUAAAAAUCCUUAUUGUUCUUGCAGUGUGCAUCGGG>hsa-mir-367 MI0000775CCAUUACUGUUGCUAAUAUGCAACUCUGUUGAAUAUAAAUUGGAAUUGCACUUUAGCAAUGGUGAUGG>hsa-mir-369 MI0000777UUGAAGGGAGAUCGACCGUGUUAUAUUCGCUUUAUUGACUUCGAAUAAUACAUGGUUGAUCUUUUCUCAG>hsa-mir-370 MI0000778AGACAGAGAAGCCAGGUCACGUCUCUGCAGUUACACAGCUCACGAGUGCCUGCUGGGGUGGAACCUGGUCUGUCU>hsa-mir-371a MI0000779GUGGCACUCAAACUGUGGGGGCACUUUCUGCUCUCUGGUGAAAGUGCCGCCAUCUUUUGAGUGUUAC>hsa-mir-371b MI0017393GGUAACACUCAAAAGAUGGCGGCACUUUCACCAGAGAGCAGAAAGUGCCCCCACAGUUUGAGUGCC>hsa-mir-372 MI0000780GUGGGCCUCAAAUGUGGAGCACUAUUCUGAUGUCCAAGUGGAAAGUGCUGCGACAUUUGAGCGUCAC>hsa-mir-373 MI0000781GGGAUACUCAAAAUGGGGGCGCUUUCCUUUUUGUCUGUACUGGGAAGUGCUUCGAUUUUGGGGUGUCCC>hsa-mir-374a MI0000782UACAUCGGCCAUUAUAAUACAACCUGAUAAGUGUUAUAGCACUUAUCAGAUUGUAUUGUAAUUGUCUGUGUA>hsa-mir-374b MI0005566ACUCGGAUGGAUAUAAUACAACCUGCUAAGUGUCCUAGCACUUAGCAGGUUGUAUUAUCAUUGUCCGUGUCU>hsa-mir-374c MI0016684ACACGGACAAUGAUAAUACAACCUGCUAAGUGCUAGGACACUUAGCAGGUUGUAUUAUAUCCAUCCGAGU>hsa-mir-375 MI0000783CCCCGCGACGAGCCCCUCGCACAAACCGGACCUGAGCGUUUUGUUCGUUCGGCUCGCGUGAGGC>hsa-mir-376a-1 MI0000784UAAAAGGUAGAUUCUCCUUCUAUGAGUACAUUAUUUAUGAUUAAUCAUAGAGGAAAAUCCACGUUUUC>hsa-mir-376a-2 MI0003529GGUAUUUAAAAGGUAGAUUUUCCUUCUAUGGUUACGUGUUUGAUGGUUAAUCAUAGAGGAAAAUCCACGUUUUCAGUAUC>hsa-mir-376b MI0002466CAGUCCUUCUUUGGUAUUUAAAACGUGGAUAUUCCUUCUAUGUUUACGUGAUUCCUGGUUAAUCAUAGAGGAAAAUCCAUGUUUUCAGUAUCAAAUGCUG>hsa-mir-376c MI0000776AAAAGGUGGAUAUUCCUUCUAUGUUUAUGUUAUUUAUGGUUAAACAUAGAGGAAAUUCCACGUUUU>hsa-mir-377 MI0000785UUGAGCAGAGGUUGCCCUUGGUGAAUUCGCUUUAUUUAUGUUGAAUCACACAAAGGCAACUUUUGUUUG>hsa-mir-378a MI0000786AGGGCUCCUGACUCCAGGUCCUGUGUGUUACCUAGAAAUAGCACUGGACUUGGAGUCAGAAGGCCU>hsa-mir-378b MI0014154GGUCAUUGAGUCUUCAAGGCUAGUGGAAAGAGCACUGGACUUGGAGGCAGAAAGACC>hsa-mir-378c MI0015825GGAGGCCAUCACUGGACUUGGAGUCAGAAGAGUGGAGUCGGGUCAGACUUCAACUCUGACUUUGAAGGUGGUGAGUGCCUC>hsa-mir-378d-1 MI0016749ACUGUUUCUGUCCUUGUUCUUGUUGUUAUUACUGGACUUGGAGUCAGAAACAGG>hsa-mir-378d-2 MI0003840GAAUGGUUACAAGGAGAGAACACUGGACUUGGAGUCAGAAAACUUUCAUCCAAGUCAUUCCCUGCUCUAAGUCCCAUUUCUGUUCCAUGAGAUUGUUU>hsa-mir-378e MI0016750CUGACUCCAGUGUCCAGGCCAGGGGCAGACAGUGGACAGAGAACAGUGCCCAAGACCACUGGACUUGGAGUCAGGACAU>hsa-mir-378f MI0016756GUCAGGUCCUGGACUCCCAUAGUUUUCAGGCUGCUAAACAACAGAACGAGCACUGGACUUGGAGCCAGAAGUCUUGGG>hsa-mir-378g MI0016761CACUGGGCUUGGAGUCAGAAGACCUGGCUCCAGCCCAGCUC>hsa-mir-378h MI0016808ACAGGAACACUGGACUUGGUGUCAGAUGGGAUGAGCCCUGGCUCUGUUUCCUAGCAGCAAUCUGAUCUUGAGCUAGUCACUGG>hsa-mir-378i MI0016902GGGAGCACUGGACUAGGAGUCAGAAGGUGGAGUUCUGGGUGCUGUUUUCCCACUCUUGGGCCCUGGGCAUGUUCUG>hsa-mir-379 MI0000787AGAGAUGGUAGACUAUGGAACGUAGGCGUUAUGAUUUCUGACCUAUGUAACAUGGUCCACUAACUCU>hsa-mir-380 MI0000788AAGAUGGUUGACCAUAGAACAUGCGCUAUCUCUGUGUCGUAUGUAAUAUGGUCCACAUCUU>hsa-mir-381 MI0000789UACUUAAAGCGAGGUUGCCCUUUGUAUAUUCGGUUUAUUGACAUGGAAUAUACAAGGGCAAGCUCUCUGUGAGUA>hsa-mir-382 MI0000790UACUUGAAGAGAAGUUGUUCGUGGUGGAUUCGCUUUACUUAUGACGAAUCAUUCACGGACAACACUUUUUUCAGUA>hsa-mir-383 MI0000791CUCCUCAGAUCAGAAGGUGAUUGUGGCUUUGGGUGGAUAUUAAUCAGCCACAGCACUGCCUGGUCAGAAAGAG>hsa-mir-384 MI0001145UGUUAAAUCAGGAAUUUUAAACAAUUCCUAGACAAUAUGUAUAAUGUUCAUAAGUCAUUCCUAGAAAUUGUUCAUAAUGCCUGUAACA>hsa-mir-409 MI0001735UGGUACUCGGGGAGAGGUUACCCGAGCAACUUUGCAUCUGGACGACGAAUGUUGCUCGGUGAACCCCUUUUCGGUAUCA>hsa-mir-410 MI0002465GGUACCUGAGAAGAGGUUGUCUGUGAUGAGUUCGCUUUUAUUAAUGACGAAUAUAACACAGAUGGCCUGUUUUCAGUACC>hsa-mir-411 MI0003675UGGUACUUGGAGAGAUAGUAGACCGUAUAGCGUACGCUUUAUCUGUGACGUAUGUAACACGGUCCACUAACCCUCAGUAUCAAAUCCAUCCCCGAG>hsa-mir-412 MI0002464CUGGGGUACGGGGAUGGAUGGUCGACCAGUUGGAAAGUAAUUGUUUCUAAUGUACUUCACCUGGUCCACUAGCCGUCCGUAUCCGCUGCAG>hsa-mir-421 MI0003685GCACAUUGUAGGCCUCAUUAAAUGUUUGUUGAAUGAAAAAAUGAAUCAUCAACAGACAUUAAUUGGGCGCCUGCUCUGUGAUCUC>hsa-mir-422a MI0001444GAGAGAAGCACUGGACUUAGGGUCAGAAGGCCUGAGUCUCUCUGCUGCAGAUGGGCUCUCUGUCCCUGAGCCAAGCUUUGUCCUCCCUGG>hsa-mir-423 MI0001445AUAAAGGAAGUUAGGCUGAGGGGCAGAGAGCGAGACUUUUCUAUUUUCCAAAAGCUCGGUCUGAGGCCCCUCAGUCUUGCUUCCUAACCCGCGC>hsa-mir-424 MI0001446CGAGGGGAUACAGCAGCAAUUCAUGUUUUGAAGUGUUCUAAAUGGUUCAAAACGUGAGGCGCUGCUAUACCCCCUCGUGGGGAAGGUAGAAGGUGGGG>hsa-mir-425 MI0001448GAAAGCGCUUUGGAAUGACACGAUCACUCCCGUUGAGUGGGCACCCGAGAAGCCAUCGGGAAUGUCGUGUCCGCCCAGUGCUCUUUC>hsa-mir-429 MI0001641CGCCGGCCGAUGGGCGUCUUACCAGACAUGGUUAGACCUGGCCCUCUGUCUAAUACUGUCUGGUAAAACCGUCCAUCCGCUGC>hsa-mir-431 MI0001721UCCUGCUUGUCCUGCGAGGUGUCUUGCAGGCCGUCAUGCAG
GCCACACUGACGGUAACGUUGCAGGUCGUCUUGCAGGGCUUCUCGCAAGACGACAUCCUCAUCACCAACGACG>hsa-mir-432 MI0003133UGACUCCUCCAGGUCUUGGAGUAGGUCAUUGGGUGGAUCCUCUAUUUCCUUACGUGGGCCACUGGAUGGCUCCUCCAUGUCUUGGAGUAGAUCA>hsa-mir-433 MI0001723CCGGGGAGAAGUACGGUGAGCCUGUCAUUAUUCAGAGAGGCUAGAUCCUCUGUGUUGAGAAGGAUCAUGAUGGGCUCCUCGGUGUUCUCCAGG>hsa-mir-448 MI0001637GCCGGGAGGUUGAACAUCCUGCAUAGUGCUGCCAGGAAAUCCCUAUUUCAUAUAAGAGGGGGCUGGCUGGUUGCAUAUGUAGGAUGUCCCAUCUCCCAGCCCACUUCGUCA>hsa-mir-449a MI0001648CUGUGUGUGAUGAGCUGGCAGUGUAUUGUUAGCUGGUUGAAUAUGUGAAUGGCAUCGGCUAACAUGCAACUGCUGUCUUAUUGCAUAUACA>hsa-mir-449b MI0003673UGACCUGAAUCAGGUAGGCAGUGUAUUGUUAGCUGGCUGCUUGGGUCAAGUCAGCAGCCACAACUACCCUGCCACUUGCUUCUGGAUAAAUUCUUCU>hsa-mir-449c MI0003823GCUGGGAUGUGUCAGGUAGGCAGUGUAUUGCUAGCGGCUGUUAAUGAUUUUAACAGUUGCUAGUUGCACUCCUCUCUGUUGCAUUCAGAAGC>hsa-mir-450a-1 MI0001652AAACGAUACUAAACUGUUUUUGCGAUGUGUUCCUAAUAUGCACUAUAAAUAUAUUGGGAACAUUUUGCAUGUAUAGUUUUGUAUCAAUAUA>hsa-mir-450a-2 MI0003187CCAAAGAAAGAUGCUAAACUAUUUUUGCGAUGUGUUCCUAAUAUGUAAUAUAAAUGUAUUGGGGACAUUUUGCAUUCAUAGUUUUGUAUCAAUAAUAUGG>hsa-mir-450b MI0005531GCAGAAUUAUUUUUGCAAUAUGUUCCUGAAUAUGUAAUAUAAGUGUAUUGGGAUCAUUUUGCAUCCAUAGUUUUGUAU>hsa-mir-451a MI0001729CUUGGGAAUGGCAAGGAAACCGUUACCAUUACUGAGUUUAGUAAUGGUAAUGGUUCUCUUGCUAUACCCAGA>hsa-mir-451b MI0017360UGGGUAUAGCAAGAGAACCAUUACCAUUACUAAACUCAGUAAUGGUAACGGUUUCCUUGCCAUUCCCA>hsa-mir-452 MI0001733GCUAAGCACUUACAACUGUUUGCAGAGGAAACUGAGACUUUGUAACUAUGUCUCAGUCUCAUCUGCAAAGAAGUAAGUGCUUUGC>hsa-mir-454 MI0003820UCUGUUUAUCACCAGAUCCUAGAACCCUAUCAAUAUUGUCUCUGCUGUGUAAAUAGUUCUGAGUAGUGCAAUAUUGCUUAUAGGGUUUUGGUGUUUGGAAAGAACAAUGGGCAGG>hsa-mir-455 MI0003513UCCCUGGCGUGAGGGUAUGUGCCUUUGGACUACAUCGUGGAAGCCAGCACCAUGCAGUCCAUGGGCAUAUACACUUGCCUCAAGGCCUAUGUCAUC>hsa-mir-466 MI0014157GUGUGUGUAUAUGUGUGUUGCAUGUGUGUAUAUGUGUGUAUAUAUGUACACAUACACAUACACGCAACACACAUAUAUACAUGC>hsa-mir-483 MI0002467GAGGGGGAAGACGGGAGGAAAGAAGGGAGUGGUUCCAUCACGCCUCCUCACUCCUCUCCUCCCGUCUUCUCCUCUC>hsa-mir-484 MI0002468AGCCUCGUCAGGCUCAGUCCCCUCCCGAUAAACCCCUAAAUAGGGACUUUCCCGGGGGGUGACCCUGGCUUUUUUGGCG>hsa-mir-485 MI0002469ACUUGGAGAGAGGCUGGCCGUGAUGAAUUCGAUUCAUCAAAGCGAGUCAUACACGGCUCUCCUCUCUUUUAGU>hsa-mir-486 MI0002470GCAUCCUGUACUGAGCUGCCCCGAGGCCCUUCAUGCUGCCCAGCUCGGGGCAGCUCAGUACAGGAUAC>hsa-mir-487a MI0002471GGUACUUGAAGAGUGGUUAUCCCUGCUGUGUUCGCUUAAUUUAUGACGAAUCAUACAGGGACAUCCAGUUUUUCAGUAUC>hsa-mir-487b MI0003530UUGGUACUUGGAGAGUGGUUAUCCCUGUCCUGUUCGUUUUGCUCAUGUCGAAUCGUACAGGGUCAUCCACUUUUUCAGUAUCAA>hsa-mir-488 MI0003123GAGAAUCAUCUCUCCCAGAUAAUGGCACUCUCAAACAAGUUUCCAAAUUGUUUGAAAGGCUAUUUCUUGGUCAGAUGACUCUC>hsa-mir-489 MI0003124GUGGCAGCUUGGUGGUCGUAUGUGUGACGCCAUUUACUUGAACCUUUAGGAGUGACAUCACAUAUACGGCAGCUAAACUGCUAC>hsa-mir-490 MI0003125UGGAGGCCUUGCUGGUUUGGAAAGUUCAUUGUUCGACACCAUGGAUCUCCAGGUGGGUCAAGUUUAGAGAUGCACCAACCUGGAGGACUCCAUGCUGUUGAGCUGUUCACAAGCAGCGGACACUUCCA>hsa-mir-491 MI0003126UUGACUUAGCUGGGUAGUGGGGAACCCUUCCAUGAGGAGUAGAACACUCCUUAUGCAAGAUUCCCUUCUACCUGGCUGGGUUGG>hsa-mir-492 MI0003131CAACUACAGCCACUACUACAGGACCAUCGAGGACCUGCGGGACAAGAUUCUUGGUGCCACCAUUGAGAACGCCAGGAUUGUCCUGCAGAUCAACAAUGCUCAACUGGCUGCAGAUG>hsa-mir-493 MI0003132CUGGCCUCCAGGGCUUUGUACAUGGUAGGCUUUCAUUCAUUCGUUUGCACAUUCGGUGAAGGUCUACUGUGUGCCAGGCCCUGUGCCAG>hsa-mir-494 MI0003134GAUACUCGAAGGAGAGGUUGUCCGUGUUGUCUUCUCUUUAUUUAUGAUGAAACAUACACGGGAAACCUCUUUUUUAGUAUC>hsa-mir-495 MI0003135UGGUACCUGAAAAGAAGUUGCCCAUGUUAUUUUCGCUUUAUAUGUGACGAAACAAACAUGGUGCACUUCUUUUUCGGUAUCA>hsa-mir-496 MI0003136CCCAAGUCAGGUACUCGAAUGGAGGUUGUCCAUGGUGUGUUCAUUUUAUUUAUGAUGAGUAUUACAUGGCCAAUCUCCUUUCGGUACUCAAUUCUUCUUGGG>hsa-mir-497 MI0003138CCACCCCGGUCCUGCUCCCGCCCCAGCAGCACACUGUGGUUUGUACGGCACUGUGGCCACGUCCAAACCACACUGUGGUGUUAGAGCGAGGGUGGGGGAGGCACCGCCGAGG>hsa-mir-498 MI0003142AACCCUCCUUGGGAAGUGAAGCUCAGGCUGUGAUUUCAAGCCAGGGGGCGUUUUUCUAUAACUGGAUGAAAAGCACCUCCAGAGCUUGAAGCUCACAGUUUGAGAGCAAUCGUCUAAGGAAGUU>hsa-mir-499a MI0003183GCCCUGUCCCCUGUGCCUUGGGCGGGCGGCUGUUAAGACUUGCAGUGAUGUUUAACUCCUCUCCACGUGAACAUCACAGCAAGUCUGUGCUGCUUCCCGUCCCUACGCUGCCUGGGCAGGGU>hsa-mir-499b MI0017396GGAAGCAGCACAGACUUGCUGUGAUGUUCACGUGGAGAGGAGUUAAACAUCACUGCAAGUCUUAACAGCCGCC>hsa-mir-500a MI0003184GCUCCCCCUCUCUAAUCCUUGCUACCUGGGUGAGAGUGCUGUCUGAAUGCAAUGCACCUGGGCAAGGAUUCUGAGAGCGAGAGC>hsa-mir-500b MI0015903CCCCCUCUCUAAUCCUUGCUACCUGGGUGAGAGUGCUUUCUGAAUGCAGUGCACCCAGGCAAGGAUUCUGCAAGGGGGA>hsa-mir-501 MI0003185GCUCUUCCUCUCUAAUCCUUUGUCCCUGGGUGAGAGUGCUUUCUGAAUGCAAUGCACCCGGGCAAGGAUUCUGAGAGGGUGAGC>hsa-mir-502 MI0003186UGCUCCCCCUCUCUAAUCCUUGCUAUCUGGGUGCUAGUGCUGGCUCAAUGCAAUGCACCUGGGCAAGGAUUCAGAGAGGGGGAGCU>hsa-mir-503 MI0003188UGCCCUAGCAGCGGGAACAGUUCUGCAGUGAGCGAUCGGUGCUCUGGGGUAUUGUUUCCGCUGCCAGGGUA>hsa-mir-504 MI0003189GCUGCUGUUGGGAGACCCUGGUCUGCACUCUAUCUGUAUUCUUACUGAAGGGAGUGCAGGGCAGGGUUUCCCAUACAGAGGGC>hsa-mir-505 MI0003190GAUGCACCCAGUGGGGGAGCCAGGAAGUAUUGAUGUUUCUGCCAGUUUAGCGUCAACACUUGCUGGUUUCCUCUCUGGAGCAUC>hsa-mir-506 MI0003193GCCACCACCAUCAGCCAUACUAUGUGUAGUGCCUUAUUCAGGAAGGUGUUACUUAAUAGAUUAAUAUUUGUAAGGCACCCUUCUGAGUAGAGUAAUGUGCAACAUGGACAACAUUUGUGGUGGC>hsa-mir-507 MI0003194GUGCUGUGUGUAGUGCUUCACUUCAAGAAGUGCCAUGCAUGUGUCUAGAAAUAUGUUUUGCACCUUUUGGAGUGAAAUAAUGCACAACAGAUAC>hsa-mir-508 MI0003195CCACCUUCAGCUGAGUGUAGUGCCCUACUCCAGAGGGCGUCACUCAUGUAAACUAAAACAUGAUUGUAGCCUUUUGGAGUAGAGUAAUACACAUCACGUAACGCAUAUUUGGUGG>hsa-mir-509-1 MI0003196CAUGCUGUGUGUGGUACCCUACUGCAGACAGUGGCAAUCAUGUAUAAUUAAAAAUGAUUGGUACGUCUGUGGGUAGAGUACUGCAUGACACAUG>hsa-mir-509-2 MI0005530CAUGCUGUGUGUGGUACCCUACUGCAGACAGUGGCAAUCAUGUAUAAUUAAAAAUGAUUGGUACGUCUGUGGGUAGAGUACUGCAUGACAC>hsa-mir-509-3 MI0005717GUGGUACCCUACUGCAGACGUGGCAAUCAUGUAUAAUUAAAAAUGAUUGGUACGUCUGUGGGUAGAGUACUGCAU>hsa-mir-510 MI0003197GUGGUGUCCUACUCAGGAGAGUGGCAAUCACAUGUAAUUAGGUGUGAUUGAAACCUCUAAGAGUGGAGUAACAC>hsa-mir-511-1 MI0003127CAAUAGACACCCAUCGUGUCUUUUGCUCUGCAGUCAGUAAAUAUUUUUUUGUGAAUGUGUAGCAAAAGACAGAAUGGUGGUCCAUUG>hsa-mir-511-2 MI0003128CAAUAGACACCCAUCGUGUCUUUUGCUCUGCAGUCAGUAAAUAUUUUUUUGUGAAUGUGUAGCAAAAGACAGAAUGGUGGUCCAUUG>hsa-mir-512-1 MI0003140UCUCAGUCUGUGGCACUCAGCCUUGAGGGCACUUUCUGGUGCCAGAAUGAAAGUGCUGUCAUAGCUGAGGUCCAAUGACUGAGG>hsa-mir-512-2 MI0003141GGUACUUCUCAGUCUGUGGCACUCAGCCUUGAGGGCACUUUCUGGUGCCAGAAUGAAAGUGCUGUCAUAGCUGAGGUCCAAUGACUGAGGCGAGCACC>hsa-mir-513a-1 MI0003191GGGAUGCCACAUUCAGCCAUUCAGCGUACAGUGCCUUUCACAGGGAGGUGUCAUUUAUGUGAACUAAAAUAUAAAUUUCACCUUUCUGAGAAGGGUAAUGUACAGCAUGCACUGCAUAUGUGGUGUCCC>hsa-mir-513a-2 MI0003192GGAUGCCACAUUCAGCCAUUCAGUGUGCAGUGCCUUUCACAGGGAGGUGUCAUUUAUGUGAACUAAAAUAUAAAUUUCACCUUUCUGAGAAGGGUAAUGUACAGCAUGCACUGCAUAUGUGGUGUCC>hsa-mir-513b MI0006648GUGUACAGUGCCUUUCACAAGGAGGUGUCAUUUAUGUGAACUAAAAUAUAAAUGUCACCUUUUUGAGAGGAGUAAUGUACAGCA>hsa-mir-513c MI0006649GCGUACAGUGCCUUUCUCAAGGAGGUGUCGUUUAUGUGAACUAAAAUAUAAAUUUCACCUUUCUGAGAAGAGUAAUGUACAGCA>hsa-mir-514a-1 MI0003198AACAUGUUGUCUGUGGUACCCUACUCUGGAGAGUGACAAUCAUGUAUAAUUAAAUUUGAUUGACACUUCUGUGAGUAGAGUAACGCAUGACACGUACG>hsa-mir-514a-2 MI0003199GUUGUCUGUGGUACCCUACUCUGGAGAGUGACAAUCAUGUAUAACUAAAUUUGAUUGACACUUCUGUGAGUAGAGUAACGCAUGACAC>hsa-mir-514a-3 MI0003200GUUGUCUGUGGUACCCUACUCUGGAGAGUGACAAUCAUGUAUAACUAAAUUUGAUUGACACUUCUGUGAGUAGAGUAACGCAUGACAC>hsa-mir-514b MI0014251CAUGUGGUACUCUUCUCAAGAGGGAGGCAAUCAUGUGUAAUUAGAUAUGAUUGACACCUCUGUGAGUGGAGUAACACAUG>hsa-mir-515-1 MI0003144UCUCAUGCAGUCAUUCUCCAAAAGAAAGCACUUUCUGUUGUCUGAAAGCAGAGUGCCUUCUUUUGGAGCGUUACUGUUUGAGA>hsa-mir-515-2 MI0003147UCUCAUGCAGUCAUUCUCCAAAAGAAAGCACUUUCUGUUGUCUGAAAGCAGAGUGCCUUCUUUUGGAGCGUUACUGUUUGAGA>hsa-mir-516a-1 MI0003180UCUCAGGCUGUGACCUUCUCGAGGAAAGAAGCACUUUCUGUUGUCUGAAAGAAAAGAAAGUGCUUCCUUUCAGAGGGUUACGGUUUGAGA>hsa-mir-516a-2 MI0003181UCUCAGGUUGUGACCUUCUCGAGGAAAGAAGCACUUUCUGUUGUCUGAAAGAAAAGAAAGUGCUUCCUUUCAGAGGGUUACGGUUUGAGA>hsa-mir-516b-1 MI0003172UCUCAGGCUGUGACCAUCUGGAGGUAAGAAGCACUUUCUGUUUUGUGAAAGAAAAGAAAGUGCUUCCUUUCAGAGGGUUACUCUUUGAGA>hsa-mir-516b-2 MI0003167UCUCAUGAUGUGACCAUCUGGAGGUAAGAAGCACUUUGUGUUUUGUGAAAGAAAGUGCUUCCUUUCAGAGGGUUACUCUUUGAGA>hsa-mir-517a MI0003161UCUCAGGCAGUGACCCUCUAGAUGGAAGCACUGUCUGUUGUAUAAAAGAAAAGAUCGUGCAUCCCUUUAGAGUGUUACUGUUUGAGA>hsa-mir-517b MI0003165GUGACCCUCUAGAUGGAAGCACUGUCUGUUGUCUAAGAAAAGAUCGUGCAUCCCUUUAGAGUGUUAC>hsa-mir-517c MI0003174GAAGAUCUCAGGCAGUGACCCUCUAGAUGGAAGCACUGUCUGUUGUCUAAGAAAAGAUCGUGCAUCCUUUUAGAGUGUUACUGUUUGAGAAAAUC>hsa-mir-518a-1 MI0003170UCUCAAGCUGUGACUGCAAAGGGAAGCCCUUUCUGUUGUCUGAAAGAAGAGAAAGCGCUUCCCUUUGCUGGAUUACGGUUUGAGA>hsa-mir-518a-2 MI0003173UCUCAAGCUGUGGGUCUGCAAAGGGAAGCCCUUUCUGUUGUCUAAAAGAAGAGAAAGCGCUUCCCUUUGCUGGAUUACGGUUUGAGA>hsa-mir-518b MI0003156UCAUGCUGUGGCCCUCCAGAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAACAAAGCGCUCCCCUUUAGAGGUUUACGGUUUGA>hsa-mir-518c MI0003159GCGAGAAGAUCUCAUGCUGUGACUCUCUGGAGGGAAGCACUUUCUGUUGUCUGAAAGAAAACAAAGCGCUUCUCUUUAGAGUGUUACGGUUUGAGAAAAGC>hsa-mir-518d MI0003171UCCCAUGCUGUGACCCUCUAGAGGGAAGCACUUUCUGUUGUCUGAAAGAAACCAAAGCGCUUCCCUUUGGAGCGUUACGGUUUGAGA>hsa-mir-518e MI0003169UCUCAGGCUGUGACCCUCUAGAGGGAAGCGCUUUCUGUUGGCUAAAAGAAAAGAAAGCGCUUCCCUUCAGAGUGUUAACGCUUUGAGA>hsa-mir-518f 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MI0003586CAUAUUAGGUUAAUGCAAAAGUAAUCGCGGUUUGUGCCAGAUGACGAUUUGAAUUAAUAAAUUCAUUUGGUAUAAACCGCGAUUAUUUUUGCAUCAAC>hsa-mir-580 MI0003587AUAAAAUUUCCAAUUGGAACCUAAUGAUUCAUCAGACUCAGAUAUUUAAGUUAACAGUAUUUGAGAAUGAUGAAUCAUUAGGUUCCGGUCAGAAAUU>hsa-mir-581 MI0003588GUUAUGUGAAGGUAUUCUUGUGUUCUCUAGAUCAGUGCUUUUAGAAAAUUUGUGUGAUCUAAAGAACACAAAGAAUACCUACACAGAACCACCUGC>hsa-mir-582 MI0003589AUCUGUGCUCUUUGAUUACAGUUGUUCAACCAGUUACUAAUCUAACUAAUUGUAACUGGUUGAACAACUGAACCCAAAGGGUGCAAAGUAGAAACAUU>hsa-mir-583 MI0003590AACUCACACAUUAACCAAAGAGGAAGGUCCCAUUACUGCAGGGAUCUUAGCAGUACUGGGACCUACCUCUUUGGU>hsa-mir-584 MI0003591UAGGGUGACCAGCCAUUAUGGUUUGCCUGGGACUGAGGAAUUUGCUGGGAUAUGUCAGUUCCAGGCCAACCAGGCUGGUUGGUCUCCCUGAAGCAAC>hsa-mir-585 MI0003592UGGGGUGUCUGUGCUAUGGCAGCCCUAGCACACAGAUACGCCCAGAGAAAGCCUGAACGUUGGGCGUAUCUGUAUGCUAGGGCUGCUGUAACAA>hsa-mir-586 MI0003594AUGGGGUAAAACCAUUAUGCAUUGUAUUUUUAGGUCCCAAUACAUGUGGGCCCUAAAAAUACAAUGCAUAAUGGUUUUUCACUCUUUAUCUUCUUAU>hsa-mir-587 MI0003595CUCCUAUGCACCCUCUUUCCAUAGGUGAUGAGUCACAGGGCUCAGGGAAUGUGUCUGCACCUGUGACUCAUCACCAGUGGAAAGCCCAUCCCAUAU>hsa-mir-588 MI0003597AGCUUAGGUACCAAUUUGGCCACAAUGGGUUAGAACACUAUUCCAUUGUGUUCUUACCCACCAUGGCCAAAAUUGGGCCUAAG>hsa-mir-589 MI0003599UCCAGCCUGUGCCCAGCAGCCCCUGAGAACCACGUCUGCUCUGAGCUGGGUACUGCCUGUUCAGAACAAAUGCCGGUUCCCAGACGCUGCCAGCUGGCC>hsa-mir-590 MI0003602UAGCCAGUCAGAAAUGAGCUUAUUCAUAAAAGUGCAGUAUGGUGAAGUCAAUCUGUAAUUUUAUGUAUAAGCUAGUCUCUGAUUGAAACAUGCAGCA>hsa-mir-591 MI0003603UCUUAUCAAUGAGGUAGACCAUGGGUUCUCAUUGUAAUAGUGUAGAAUGUUGGUUAACUGUGGACUCCCUGGCUCUGUCUCAAAUCUACUGAUUC>hsa-mir-592 MI0003604UAUUAUGCCAUGACAUUGUGUCAAUAUGCGAUGAUGUGUUGUGAUGGCACAGCGUCAUCACGUGGUGACGCAACAUCAUGACGUAAGACGUCACAAC>hsa-mir-593 MI0003605CCCCCAGAAUCUGUCAGGCACCAGCCAGGCAUUGCUCAGCCCGUUUCCCUCUGGGGGAGCAAGGAGUGGUGCUGGGUUUGUCUCUGCUGGGGUUUCUCCU>hsa-mir-595 MI0003607ACGGAAGCCUGCACGCAUUUAACACCAGCACGCUCAAUGUAGUCUUGUAAGGAACAGGUUGAAGUGUGCCGUGGUGUGUCUGGAGGAAGCGCCUGU>hsa-mir-596 MI0003608AGCACGGCCUCUCCGAAGCCUGCCCGGCUCCUCGGGAACCUGCCUCCCGCAUGGCAGCUGCUGCCCUUCGGAGGCCG>hsa-mir-597 MI0003609UACUUACUCUACGUGUGUGUCACUCGAUGACCACUGUGAAGACAGUAAAAUGUACAGUGGUUCUCUUGUGGCUCAAGCGUAAUGUAGAGUACUGGUC>hsa-mir-598 MI0003610GCUUGAUGAUGCUGCUGAUGCUGGCGGUGAUCCCGAUGGUGUGAGCUGGAAAUGGGGUGCUACGUCAUCGUUGUCAUCGUCAUCAUCAUCAUCCGAG>hsa-mir-599 MI0003611AAAGACAUGCUGUCCACAGUGUGUUUGAUAAGCUGACAUGGGACAGGGAUUCUUUUCACUGUUGUGUCAGUUUAUCAAACCCAUACUUGGAUGAC>hsa-mir-600 MI0003613AAGUCACGUGCUGUGGCUCCAGCUUCAUAGGAAGGCUCUUGUCUGUCAGGCAGUGGAGUUACUUACAGACAAGAGCCUUGCUCAGGCCAGCCCUGCCC>hsa-mir-601 MI0003614UGCAUGAGUUCGUCUUGGUCUAGGAUUGUUGGAGGAGUCAGAAAAACUACCCCAGGGAUCCUGAAGUCCUUUGGGUGGA>hsa-mir-602 MI0003615UUCUCACCCCCGCCUGACACGGGCGACAGCUGCGGCCCGCUGUGUUCACUCGGGCCGAGUGCGUCUCCUGUCAGGCAAGGGAGAGCAGAGCCCCCCUG>hsa-mir-603 MI0003616GAUUGAUGCUGUUGGUUUGGUGCAAAAGUAAUUGCAGUGCUUCCCAUUUAAAAGUAAUGGCACACACUGCAAUUACUUUUGCUCCAACUUAAUACUU>hsa-mir-604 MI0003617AGAGCAUCGUGCUUGACCUUCCACGCUCUCGUGUCCACUAGCAGGCAGGUUUUCUGACACAGGCUGCGGAAUUCAGGACAGUGCAUCAUGGAGA>hsa-mir-605 MI0003618GCCCUAGCUUGGUUCUAAAUCCCAUGGUGCCUUCUCCUUGGGAAAAACAGAGAAGGCACUAUGAGAUUUAGAAUCAAGUUAGG>hsa-mir-606 MI0003619UGUAUCCUUGGUUUUUAGUAGUUUUACUAUGAUGAGGUGUGCCAUCCACCCCAUCAUAGUAAACUACUGAAAAUCAAAGAUACAAGUGCCUGACCA>hsa-mir-607 MI0003620UUGCCUAAAGUCACACAGGUUAUAGAUCUGGAUUGGAACCCAGGGAGCCAGACUGCCUGGGUUCAAAUCCAGAUCUAUAACUUGUGUGACUUUGGG>hsa-mir-608 MI0003621GGGCCAAGGUGGGCCAGGGGUGGUGUUGGGACAGCUCCGUUUAAAAAGGCAUCUCCAAGAGCUUCCAUCAAAGGCUGCCUCUUGGUGCAGCACAGGUAGA>hsa-mir-609 MI0003622UGCUCGGCUGUUCCUAGGGUGUUUCUCUCAUCUCUGGUCUAUAAUGGGUUAAAUAGUAGAGAUGAGGGCAACACCCUAGGAACAGCAGAGGAACC>hsa-mir-610 MI0003623UCUAUUUGUCUUAGGUGAGCUAAAUGUGUGCUGGGACACAUUUGAGCCAAAUGUCCCAGCACACAUUUAGCUCACAUAAGAAAAAUGGACUCUAGU>hsa-mir-611 MI0003624AAAAUGGUGAGAGCGUUGAGGGGAGUUCCAGACGGAGAUGCGAGGACCCCUCGGGGUCUGACCCACA>hsa-mir-612 MI0003625UCCCAUCUGGACCCUGCUGGGCAGGGCUUCUGAGCUCCUUAGCACUAGCAGGAGGGGCUCCAGGGGCCCUCCCUCCAUGGCAGCCAGGACAGGACUCUCA>hsa-mir-613 MI0003626GGUGAGUGCGUUUCCAAGUGUGAAGGGACCCUUCCUGUAGUGUCUUAUAUACAAUACAGUAGGAAUGUUCCUUCUUUGCCACUCAUACACCUUUA>hsa-mir-614 MI0003627UCUAAGAAACGCAGUGGUCUCUGAAGCCUGCAGGGGCAGGCCAGCCCUGCACUGAACGCCUGUUCUUGCCAGGUGGCAGAAGGUUGCUGC>hsa-mir-615 MI0003628CUCGGGAGGGGCGGGAGGGGGGUCCCCGGUGCUCGGAUCUCGAGGGUGCUUAUUGUUCGGUCCGAGCCUGGGUCUCCCUCUUCCCCCCAACCCCCC>hsa-mir-616 MI0003629UUAGGUAAUUCCUCCACUCAAAACCCUUCAGUGACUUCCAUGACAUGAAAUAGGAAGUCAUUGGAGGGUUUGAGCAGAGGAAUGACCUGUUUUAAAA>hsa-mir-617 MI0003631CAUCAUAAGGAGCCUAGACUUCCCAUUUGAAGGUGGCCAUUUCCUACCACCUUCAAAUGGUAAGUCCAGGCUCCUUCUGAUUCAAUAAAUGAGGAGC>hsa-mir-618 MI0003632CUCUUGUUCACAGCCAAACUCUACUUGUCCUUCUGAGUGUAAUUACGUACAUGCAGUAGCUCAGGAGACAAGCAGGUUUACCCUGUGGAUGAGUCUGA>hsa-mir-619 MI0003633CGCCCACCUCAGCCUCCCAAAAUGCUGGGAUUACAGGCAUGAGCCACUGCGGUCGACCAUGACCUGGACAUGUUUGUGCCCAGUACUGUCAGUUUGCAG>hsa-mir-620 MI0003634AUAUAUAUCUAUAUCUAGCUCCGUAUAUAUAUAUAUAUAUAUAUAGAUAUCUCCAUAUAUAUGGAGAUAGAUAUAGAAAUAAAACAAGCAAAGAA>hsa-mir-621 MI0003635UAGAUUGAGGAAGGGGCUGAGUGGUAGGCGGUGCUGCUGUGCUCUGAUGAAGACCCAUGUGGCUAGCAACAGCGCUUACCUUUUGUCUCUGGGUCC>hsa-mir-622 MI0003636AGAGAAGCUGGACAAGUACUGGUCUCAGCAGAUUGAGGAGAGCACCACAGUGGUCAUCACACAGUCUGCUGAGGUUGGAGCUGCUGAGAUGACACU>hsa-mir-623 MI0003637GUACACAGUAGAAGCAUCCCUUGCAGGGGCUGUUGGGUUGCAUCCUAAGCUGUGCUGGAGCUUCCCGAUGUACUCUGUAGAUGUCUUUGCACCUUCUG>hsa-mir-624 MI0003638AAUGCUGUUUCAAGGUAGUACCAGUACCUUGUGUUCAGUGGAACCAAGGUAAACACAAGGUAUUGGUAUUACCUUGAGAUAGCAUUACACCUAAGUG>hsa-mir-625 MI0003639AGGGUAGAGGGAUGAGGGGGAAAGUUCUAUAGUCCUGUAAUUAGAUCUCAGGACUAUAGAACUUUCCCCCUCAUCCCUCUGCCCU>hsa-mir-626 MI0003640ACUGAUAUAUUUGUCUUAUUUGAGAGCUGAGGAGUAUUUUUAUGCAAUCUGAAUGAUCUCAGCUGUCUGAAAAUGUCUUCAAUUUUAAAGGCUU>hsa-mir-627 MI0003641UACUUAUUACUGGUAGUGAGUCUCUAAGAAAAGAGGAGGUGGUUGUUUUCCUCCUCUUUUCUUUGAGACUCACUACCAAUAAUAAGAAAUACUACUA>hsa-mir-628 MI0003642AUAGCUGUUGUGUCACUUCCUCAUGCUGACAUAUUUACUAGAGGGUAAAAUUAAUAACCUUCUAGUAAGAGUGGCAGUCGAAGGGAAGGGCUCAU>hsa-mir-629 MI0003643UCCCUUUCCCAGGGGAGGGGCUGGGUUUACGUUGGGAGAACUUUUACGGUGAACCAGGAGGUUCUCCCAACGUAAGCCCAGCCCCUCCCCUCUGCCU>hsa-mir-630 MI0003644AACUUAACAUCAUGCUACCUCUUUGUAUCAUAUUUUGUUAUUCUGGUCACAGAAUGACCUAGUAUUCUGUACCAGGGAAGGUAGUUCUUAACUAUAU>hsa-mir-631 MI0003645GUGGGGAGCCUGGUUAGACCUGGCCCAGACCUCAGCUACACAAGCUGAUGGACUGAGUCAGGGGCCACACUCUCC>hsa-mir-632 MI0003647CGCCUCCUACCGCAGUGCUUGACGGGAGGCGGAGCGGGGAACGAGGCCGUCGGCCAUUUUGUGUCUGCUUCCUGUGGGACGUGGUGGUAGCCGU>hsa-mir-633 MI0003648AACCUCUCUUAGCCUCUGUUUCUUUAUUGCGGUAGAUACUAUUAACCUAAAAUGAGAAGGCUAAUAGUAUCUACCACAAUAAAAUUGUUGUGAGGAUA>hsa-mir-634 MI0003649AAACCCACACCACUGCAUUU
UGGCCAUCGAGGGUUGGGGCUUGGUGUCAUGCCCCAAGAUAACCAGCACCCCAACUUUGGACAGCAUGGAUUAGUCU>hsa-mir-635 MI0003650CAGAGAGGAGCUGCCACUUGGGCACUGAAACAAUGUCCAUUAGGCUUUGUUAUGGAAACUUCUCCUGAUCAUUGUUUUGUGUCCAUUGAGCUUCCAAU>hsa-mir-636 MI0003651UGGCGGCCUGGGCGGGAGCGCGCGGGCGGGGCCGGCCCCGCUGCCUGGAAUUAACCCCGCUGUGCUUGCUCGUCCCGCCCGCAGCCCUAGGCGGCGUCG>hsa-mir-637 MI0003652UGGCUAAGGUGUUGGCUCGGGCUCCCCACUGCAGUUACCCUCCCCUCGGCGUUACUGAGCACUGGGGGCUUUCGGGCUCUGCGUCUGCACAGAUACUUC>hsa-mir-638 MI0003653GUGAGCGGGCGCGGCAGGGAUCGCGGGCGGGUGGCGGCCUAGGGCGCGGAGGGCGGACCGGGAAUGGCGCGCCGUGCGCCGCCGGCGUAACUGCGGCGCU>hsa-mir-639 MI0003654UGGCCGACGGGGCGCGCGCGGCCUGGAGGGGCGGGGCGGACGCAGAGCCGCGUUUAGUCUAUCGCUGCGGUUGCGAGCGCUGUAGGGAGCCUGUGCUG>hsa-mir-640 MI0003655GUGACCCUGGGCAAGUUCCUGAAGAUCAGACACAUCAGAUCCCUUAUCUGUAAAAUGGGCAUGAUCCAGGAACCUGCCUCUACGGUUGCCUUGGGG>hsa-mir-641 MI0003656UGGGUGAAAGGAAGGAAAGACAUAGGAUAGAGUCACCUCUGUCCUCUGUCCUCUACCUAUAGAGGUGACUGUCCUAUGUCUUUCCUUCCUCUUACCCCU>hsa-mir-642a MI0003657AUCUGAGUUGGGAGGGUCCCUCUCCAAAUGUGUCUUGGGGUGGGGGAUCAAGACACAUUUGGAGAGGGAACCUCCCAACUCGGCCUCUGCCAUCAUU>hsa-mir-642b MI0016685GAGUUGGGAGGUUCCCUCUCCAAAUGUGUCUUGAUCCCCCACCCCAAGACACAUUUGGAGAGGGACCCUCCCAACUC>hsa-mir-643 MI0003658ACCAAGUGAUAUUCAUUGUCUACCUGAGCUAGAAUACAAGUAGUUGGCGUCUUCAGAGACACUUGUAUGCUAGCUCAGGUAGAUAUUGAAUGAAAAA>hsa-mir-644a MI0003659UUUUUUUUUAGUAUUUUUCCAUCAGUGUUCAUAAGGAAUGUUGCUCUGUAGUUUUCUUAUAGUGUGGCUUUCUUAGAGCAAAGAUGGUUCCCUA>hsa-mir-644b MI0019134UGGGCUAAGGGAGAUGAUUGGGUAGAAAGUAUUAUUCUAUUCAUUUGCCUCCCAGCCUACA>hsa-mir-645 MI0003660CAGUUCCUAACAGGCCUCAGACCAGUACCGGUCUGUGGCCUGGGGGUUGAGGACCCCUGCUCUAGGCUGGUACUGCUGAUGCUUAAAAAGAGAG>hsa-mir-646 MI0003661GAUCAGGAGUCUGCCAGUGGAGUCAGCACACCUGCUUUUCACCUGUGAUCCCAGGAGAGGAAGCAGCUGCCUCUGAGGCCUCAGGCUCAGUGGC>hsa-mir-647 MI0003662AGGAAGUGUUGGCCUGUGGCUGCACUCACUUCCUUCAGCCCCAGGAAGCCUUGGUCGGGGGCAGGAGGGAGGGUCAGGCAGGGCUGGGGGCCUGAC>hsa-mir-648 MI0003663AUCACAGACACCUCCAAGUGUGCAGGGCACUGGUGGGGGCCGGGGCAGGCCCAGCGAAAGUGCAGGACCUGGCACUUAGUCGGAAGUGAGGGUG>hsa-mir-649 MI0003664GGCCUAGCCAAAUACUGUAUUUUUGAUCGACAUUUGGUUGAAAAAUAUCUAUGUAUUAGUAAACCUGUGUUGUUCAAGAGUCCACUGUGUUUUGCUG>hsa-mir-650 MI0003665CAGUGCUGGGGUCUCAGGAGGCAGCGCUCUCAGGACGUCACCACCAUGGCCUGGGCUCUGCUCCUCCUCACCCUCCUCACUCAGGGCACAGGUGAU>hsa-mir-651 MI0003666AAUCUAUCACUGCUUUUUAGGAUAAGCUUGACUUUUGUUCAAAUAAAAAUGCAAAAGGAAAGUGUAUCCUAAAAGGCAAUGACAGUUUAAUGUGUUU>hsa-mir-652 MI0003667ACGAAUGGCUAUGCACUGCACAACCCUAGGAGAGGGUGCCAUUCACAUAGACUAUAAUUGAAUGGCGCCACUAGGGUUGUGCAGUGCACAACCUACAC>hsa-mir-653 MI0003674UUCAUUCCUUCAGUGUUGAAACAAUCUCUACUGAACCAGCUUCAAACAAGUUCACUGGAGUUUGUUUCAAUAUUGCAAGAAUGAUAAGAUGGAAGC>hsa-mir-654 MI0003676GGGUAAGUGGAAAGAUGGUGGGCCGCAGAACAUGUGCUGAGUUCGUGCCAUAUGUCUGCUGACCAUCACCUUUAGAAGCCC>hsa-mir-655 MI0003677AACUAUGCAAGGAUAUUUGAGGAGAGGUUAUCCGUGUUAUGUUCGCUUCAUUCAUCAUGAAUAAUACAUGGUUAACCUCUUUUUGAAUAUCAGACUC>hsa-mir-656 MI0003678CUGAAAUAGGUUGCCUGUGAGGUGUUCACUUUCUAUAUGAUGAAUAUUAUACAGUCAACCUCUUUCCGAUAUCGAAUC>hsa-mir-657 MI0003681GUGUAGUAGAGCUAGGAGGAGAGGGUCCUGGAGAAGCGUGGACCGGUCCGGGUGGGUUCCGGCAGGUUCUCACCCUCUCUAGGCCCCAUUCUCCUCUG>hsa-mir-658 MI0003682GCUCGGUUGCCGUGGUUGCGGGCCCUGCCCGCCCGCCAGCUCGCUGACAGCACGACUCAGGGCGGAGGGAAGUAGGUCCGUUGGUCGGUCGGGAACGAGG>hsa-mir-659 MI0003683UACCGACCCUCGAUUUGGUUCAGGACCUUCCCUGAACCAAGGAAGAGUCACAGUCUCUUCCUUGGUUCAGGGAGGGUCCCCAACAAUGUCCUCAUGG>hsa-mir-660 MI0003684CUGCUCCUUCUCCCAUACCCAUUGCAUAUCGGAGUUGUGAAUUCUCAAAACACCUCCUGUGUGCAUGGAUUACAGGAGGGUGAGCCUUGUCAUCGUG>hsa-mir-661 MI0003669GGAGAGGCUGUGCUGUGGGGCAGGCGCAGGCCUGAGCCCUGGUUUCGGGCUGCCUGGGUCUCUGGCCUGCGCGUGACUUUGGGGUGGCU>hsa-mir-662 MI0003670GCUGUUGAGGCUGCGCAGCCAGGCCCUGACGGUGGGGUGGCUGCGGGCCUUCUGAAGGUCUCCCACGUUGUGGCCCAGCAGCGCAGUCACGUUGC>hsa-mir-663a MI0003672CCUUCCGGCGUCCCAGGCGGGGCGCCGCGGGACCGCCCUCGUGUCUGUGGCGGUGGGAUCCCGCGGCCGUGUUUUCCUGGUGGCCCGGCCAUG>hsa-mir-663b MI0006336GGUGCCGAGGGCCGUCCGGCAUCCUAGGCGGGUCGCUGCGGUACCUCCCUCCUGUCUGUGGCGGUGGGAUCCCGUGGCCGUGUUUUCCUGGUGGCCCGGCCGUGCCUGAGGUUUC>hsa-mir-664 MI0006442GAACAUUGAAACUGGCUAGGGAAAAUGAUUGGAUAGAAACUAUUAUUCUAUUCAUUUAUCCCCAGCCUACAAAAUGAAAAAA>hsa-mir-665 MI0005563UCUCCUCGAGGGGUCUCUGCCUCUACCCAGGACUCUUUCAUGACCAGGAGGCUGAGGCCCCUCACAGGCGGC>hsa-mir-668 MI0003761GGUAAGUGCGCCUCGGGUGAGCAUGCACUUAAUGUGGGUGUAUGUCACUCGGCUCGGCCCACUACC>hsa-mir-670 MI0003933GUUUAGGGGUGGACCUGAUGUCCCUGAGUGUAUGUGGUGAACCUGAAUUUGCCUUGGGUUUCCUCAUAUUCAUUCAGGAGUGUCAGUUGCCCCUUCAC>hsa-mir-671 MI0003760GCAGGUGAACUGGCAGGCCAGGAAGAGGAGGAAGCCCUGGAGGGGCUGGAGGUGAUGGAUGUUUUCCUCCGGUUCUCAGGGCUCCACCUCUUUCGGGCCGUAGAGCCAGGGCUGGUGC>hsa-mir-675 MI0005416CCCAGGGUCUGGUGCGGAGAGGGCCCACAGUGGACUUGGUGACGCUGUAUGCCCUCACCGCUCAGCCCCUGGG>hsa-mir-676 MI0016436GCAUGACUCUUCAACCUCAGGACUUGCAGAAUUAAUGGAAUGCUGUCCUAAGGUUGUUGAGUUGUGC>hsa-mir-708 MI0005543AACUGCCCUCAAGGAGCUUACAAUCUAGCUGGGGGUAAAUGACUUGCACAUGAACACAACUAGACUGUGAGCUUCUAGAGGGCAGGGA>hsa-mir-711 MI0012488ACUGACUUUGAGUCUCUCCUCAGGGUGCUGCAGGCAAAGCUGGGGACCCAGGGAGAGACGUAAGUGAGGGGAGAUG>hsa-mir-718 MI0012489GGCCGCGGCGCGCAAGAUGGCGGCGGGCCCGGGCACCGCCCCUUCCGCCCCGCCGGGCGUCGCACGAGGC>hsa-mir-720 MI0006654CCGGAUCUCACACGGUGGUGUUAAUAUCUCGCUGGGGCCUCCAAAAUGUUGUGCCCAGGGGUGUUAGAGAAAACACCACACUUUGAGAUGAAUUAAGAGUCCUUUAUUAG>hsa-mir-744 MI0005559UUGGGCAAGGUGCGGGGCUAGGGCUAACAGCAGUCUUACUGAAGGUUUCCUGGAAACCACGCACAUGCUGUUGCCACUAACCUCAACCUUACUCGGUC>hsa-mir-758 MI0003757GCCUGGAUACAUGAGAUGGUUGACCAGAGAGCACACGCUUUAUUUGUGCCGUUUGUGACCUGGUCCACUAACCCUCAGUAUCUAAUGC>hsa-mir-759 MI0004065UAAUAAAUUAAAUGCCUAAACUGGCAGAGUGCAAACAAUUUUGACUCAGAUCUAAAUGUUUGCACUGGCUGUUUAAACAUUUAAUUUGUUA>hsa-mir-760 MI0005567GGCGCGUCGCCCCCCUCAGUCCACCAGAGCCCGGAUACCUCAGAAAUUCGGCUCUGGGUCUGUGGGGAGCGAAAUGCAAC>hsa-mir-761 MI0003941GGAGGAGCAGCAGGGUGAAACUGACACAGUUCUGGUGAGUUUCACUUUGCUGCUCCUCC>hsa-mir-762 MI0003892GGCCCGGCUCCGGGUCUCGGCCCGUACAGUCCGGCCGGCCAUGCUGGCGGGGCUGGGGCCGGGGCCGAGCCCGCGGCGGGGCC>hsa-mir-764 MI0003944AAUCUAGGAGGCAGGUGCUCACUUGUCCUCCUCCAUGCUUGGAAAAUGCAGGGAGGAGGCCAUAGUGGCAACUGUUACCAUGAUU>hsa-mir-765 MI0005116UUUAGGCGCUGAUGAAAGUGGAGUUCAGUAGACAGCCCUUUUCAAGCCCUACGAGAAACUGGGGUUUCUGGAGGAGAAGGAAGGUGAUGAAGGAUCUGUUCUCGUGAGCCUGAA>hsa-mir-766 MI0003836GCAUCCUCAGGACCUGGGCUUGGGUGGUAGGAGGAAUUGGUGCUGGUCUUUCAUUUUGGAUUUGACUCCAGCCCCACAGCCUCAGCCACCCCAGCCAAUUGUCAUAGGAGC>hsa-mir-767 MI0003763GCUUUUAUAUUGUAGGUUUUUGCUCAUGCACCAUGGUUGUCUGAGCAUGCAGCAUGCUUGUCUGCUCAUACCCCAUGGUUUCUGAGCAGGAACCUUCAUUGUCUACUGC>hsa-mir-769 MI0003834GCCUUGGUGCUGAUUCCUGGGCUCUGACCUGAGACCUCUGGGUUCUGAGCUGUGAUGUUGCUCUCGAGCUGGGAUCUCCGGGGUCUUGGUUCAGGGCCGGGGCCUCUGGGUUCCAAGC>hsa-mir-770 MI0005118AGGAGCCACCUUCCGAGCCUCCAGUACCACGUGUCAGGGCCACAUGAGCUGGGCCUCGUGGGCCUGAUGUGGUGCUGGGGCCUCAGGGGUCUGCUCUU>hsa-mir-802 MI0003906GUUCUGUUAUUUGCAGUCAGUAACAAAGAUUCAUCCUUGUGUCCAUCAUGCAACAAGGAGAAUCUUUGUCACUUAGUGUAAUUAAUAGCUGGAC>hsa-mir-873 MI0005564GUGUGCAUUUGCAGGAACUUGUGAGUCUCCUAUUGAAAAUGAACAGGAGACUGAUGAGUUCCCGGGAACACCCACAA>hsa-mir-874 MI0005532UUAGCCCUGCGGCCCCACGCACCAGGGUAAGAGAGACUCUCGCUUCCUGCCCUGGCCCGAGGGACCGACUGGCUGGGC>hsa-mir-875 MI0005541UUAGUGGUACUAUACCUCAGUUUUAUCAGGUGUUCUUAAAAUCACCUGGAAACACUGAGGUUGUGUCUCACUGAAC>hsa-mir-876 MI0005542UGAAGUGCUGUGGAUUUCUUUGUGAAUCACCAUAUCUAAGCUAAUGUGGUGGUGGUUUACAAAGUAAUUCAUAGUGCUUCA>hsa-mir-877 MI0005561GUAGAGGAGAUGGCGCAGGGGACACGGGCAAAGACUUGGGGGUUCCUGGGACCCUCAGACGUGUGUCCUCUUCUCCCUCCUCCCAG>hsa-mir-885 MI0005560CCGCACUCUCUCCAUUACACUACCCUGCCUCUUCUCCAUGAGAGGCAGCGGGGUGUAGUGGAUAGAGCACGGGU>hsa-mir-887 MI0005562GUGCAGAUCCUUGGGAGCCCUGUUAGACUCUGGAUUUUACACUUGGAGUGAACGGGCGCCAUCCCGAGGCUUUGCACAG>hsa-mir-888 MI0005537GGCAGUGCUCUACUCAAAAAGCUGUCAGUCACUUAGAUUACAUGUGACUGACACCUCUUUGGGUGAAGGAAGGCUCA>hsa-mir-889 MI0005540GUGCUUAAAGAAUGGCUGUCCGUAGUAUGGUCUCUAUAUUUAUGAUGAUUAAUAUCGGACAACCAUUGUUUUAGUAUCC>hsa-mir-890 MI0005533GGAAGUGCCCUACUUGGAAAGGCAUCAGUUGCUUAGAUUACAUGUAACUAUUCCCUUUCUGAGUAGAGUAAGUCUUA>hsa-mir-891a MI0005524CCUUAAUCCUUGCAACGAACCUGAGCCACUGAUUCAGUAAAAUACUCAGUGGCACAUGUUUGUUGUGAGGGUCAAAAGA>hsa-mir-891b MI0005534CCUUAAUCCUUGCAACUUACCUGAGUCAUUGAUUCAGUAAAACAUUCAAUGGCACAUGUUUGUUGUUAGGGUCAAAAGA>hsa-mir-892a MI0005528GCAGUGCCUUACUCAGAAAGGUGCCAGUCACUUACACUACAUGUCACUGUGUCCUUUCUGCGUAGAGUAAGGCUC>hsa-mir-892b MI0005538UGCAAUGCCCUACUCAGAAAGGUGCCAUUUAUGUAGAUUUUAUGUCACUGGCUCCUUUCUGGGUAGAGCAAGGCUCA>hsa-mir-920 MI0005712GUAGUUGUUCUACAGAAGACCUGGAUGUGUAGGAGCUAAGACACACUCCAGGGGAGCUGUGGAAGCAGUAACACG>hsa-mir-921 MI0005713ACUAGUGAGGGACAGAACCAGGAUUCAGACUCAGGUCCAUGGGCCUGGAUCACUGG>hsa-mir-922 MI0005714AUGGCGUUUUCCCUCUCCCUGUCCUGGACUGGGGUCAGACUGUGCCCCGAGGAGAAGCAGCAGAGAAUAGGACUACGUCAU>hsa-mir-924 MI0005716AAUAGAGUCUUGUGAUGUCUUGCUUAAGGGCCAUCCAACCUAGAGUCUACAAC>hsa-mir-933 MI0005755ACUUGGGUCAGUUCAGAGGUCCUCGGGGCGCGCGUCGAGUCAGCCGUGUGCGCAGGGAGACCUCUCCCACCCACAGU>hsa-mir-934 MI0005756AGAAAUAAGGCUUCUGUCUACUACUGGAGACACUGGUAGUAUAAAACCCAGAGUCUCCAGUAAUGGACGGGAGCCUUAUUUCU>hsa-mir-935 MI0005757GGCGGGGGCGCGGGCGGCAGUGGCGGGAGCGGCCCCUCGGCCAUCCUCCGUCUGCCCAGUUACCGCUUCCGCUACCGCCGCCGCUCCCGCU>hsa-mir-936 MI0005758UCAAGGCCACUGGGACAGUAGAGGGAGGAAUCGCAGAAAUCACUCCAGGAGCAACUGAGAGACCUUGCUUCUACUUUACCAGGUCCUGCUGGCCCAGA>hsa-mir-937 MI0005759AGCACUGCCCCCGGUGAGUCAGGGUGGGGCUGGCCCCCUGCUUCGUGCCCAUCCGCGCUCUGACUCUCUGCCCACCUGCAGGAGCU>hsa-mir-938 MI0005760GAAGGUGUACCAUGUGCCCUUAAAGGUGAACCCAGUGCACCUUCAUGAACCGUGGUACACCUUUAAGAACUUGGUAUGCCUUC>hsa-mir-939 MI0005761UGUGGGCAGGGCCCUGGGGAGCUGAGGCUCUGGGGGUGGCCGGGGCUGACCCUGGGCCUCUGCUCCCCAGUGUCUGACCGCG>hsa-mir-940 MI0005762GUGAGGUGUGGGCCCGGCCCCAGGAGCGGGGCCUGGGCAGCCCCGUGUGUUGAGGAAGGAAGGCAGGGCCCCCGCUCCCCGGGCCUGACCCCAC>hsa-mir-941-1 MI0005763CACGGAAGAGGACACACCCGGCUGUGUGGACAUGUGCCCAGGGCCCGGGACAGCGCCACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGG>hsa-mir-941-2 MI0005764CACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGG>hsa-mir-941-3 MI0005765CACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCAUGG>hsa-mir-941-4 MI0005766CAUGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGG>hsa-mir-942 MI0005767AUUAGGAGAGUAUCUUCUCUGUUUUGGCCAUGUGUGUACUCACAGCCCCUCACACAUGGCCGAAACAGAGAAGUUACUUUCCUAAU>hsa-mir-943 MI0005768GGGACGUUCUGAGCUCGGGGUGGGGGACGUUUGCCGGUCACUGCUGCUGGCGCCCUGACUGUUGCCGUCCUCCAGCCCCACUCAAAGGCAUCCC>hsa-mir-944 MI0005769GUUCCAGACACAUCUCAUCUGAUAUACAAUAUUUUCUUAAAUUGUAUAAAGAGAA
AUUAUUGUACAUCGGAUGAGCUGUGUCUGGGAU>hsa-mir-1178 MI0006271GCGUUGGCUGGCAGAGGAAGGGAAGGGUCCAGGGUCAGCUGAGCAUGCCCUCAGGUUGCUCACUGUUCUUCCCUAGAAUGUCAGGUGAUGU>hsa-mir-1179 MI0006272GGCUGGAAAGGAAGAAGCAUUCUUUCAUUGGUUGGUGUGUAUUGCCUUGUCAACCAAUAAGAGGAUGCCAUUUAUCCUUUUCUGACUAGCU>hsa-mir-1180 MI0006273GCUGCUGGACCCACCCGGCCGGGAAUAGUGCUCCUGGUUGUUUCCGGCUCGCGUGGGUGUGUCGGCGGC>hsa-mir-1181 MI0006274UCCACUGCUGCCGCCGUCGCCGCCACCCGAGCCGGAGCGGGCUGGGCCGCCAAGGCAAGAUGGUGGACUACAGCGUGUGGG>hsa-mir-1182 MI0006275GGGACUUGUCACUGCCUGUCUCCUCCCUCUCCAGCAGCGACUGGAUUCUGGAGUCCAUCUAGAGGGUCUUGGGAGGGAUGUGACUGUUGGGAAGCCC>hsa-mir-1183 MI0006276AUUAUUCAAAUGCUCGGAGACACAGAACAUUAGAGAAGACAGGAGUUCACUGUAGGUGAUGGUGAGAGUGGGCAUGGAGCAGGAGUGCC>hsa-mir-1184-1 MI0006277CUUGCAGAACGAGGUGAAGGAGGUGGUUCUGCUCAGCAGUCAACAGUGGCCACAUCUCCACCUGCAGCGACUUGAUGGCUUCCGUGUCCUUUUCGUGGG>hsa-mir-1184-2 MI0015971CUUGCAGAACGAGGUGAAGGAGGUGGUUCUGCUCAGCAGUCAACAGUGGCCACAUCUCCACCUGCAGCGACUUGAUGGCUUCCGUGUCCUUUUCGUGGG>hsa-mir-1184-3 MI0015972CUUGCAGAACGAGGUGAAGGAGGUGGUUCUGCUCAGCAGUCAACAGUGGCCACAUCUCCACCUGCAGCGACUUGAUGGCUUCCGUGUCCUUUUCGUGGG>hsa-mir-1185-1 MI0003844UUUGGUACUUGAAGAGAGGAUACCCUUUGUAUGUUCACUUGAUUAAUGGCGAAUAUACAGGGGGAGACUCUUAUUUGCGUAUCAAA>hsa-mir-1185-2 MI0003821UUUGGUACUUAAAGAGAGGAUACCCUUUGUAUGUUCACUUGAUUAAUGGCGAAUAUACAGGGGGAGACUCUCAUUUGCGUAUCAAA>hsa-mir-1193 MI0014205GUAGCUGAGGGGAUGGUAGACCGGUGACGUGCACUUCAUUUACGAUGUAGGUCACCCGUUUGACUAUCCACCAGCGCC>hsa-mir-1197 MI0006656ACUUCCUGGUAUUUGAAGAUGCGGUUGACCAUGGUGUGUACGCUUUAUUUGUGACGUAGGACACAUGGUCUACUUCUUCUCAAUAUCA>hsa-mir-1200 MI0006332UGCUACUUCUCCUGAGCCAUUCUGAGCCUCAAUCACUUGCCAGAGAGAUUGGUUCAGGAAUUUGUCAGGGAUAGCC>hsa-mir-1202 MI0006334CCUGCUGCAGAGGUGCCAGCUGCAGUGGGGGAGGCACUGCCAGGGCUGCCCACUCUGCUUAGCCAGCAGGUGCCAAGAACAGG>hsa-mir-1203 MI0006335UCCUCCCCGGAGCCAGGAUGCAGCUCAAGCCACAGCAGGGUGUUUAGCGCUCUUCAGUGGCUCCAGAUUGUGGCGCUGGUGCAGG>hsa-mir-1204 MI0006337ACCUCGUGGCCUGGUCUCCAUUAUUUGAGAUGAGUUACAUCUUGGAGGUGAGGACGUGCCUCGUGGU>hsa-mir-1205 MI0006338GAAGGCCUCUGCAGGGUUUGCUUUGAGGUACUUCCUUCCUGUCAACCCUGUUCUGGAGUCUGU>hsa-mir-1206 MI0006339CAGUGUUCAUGUAGAUGUUUAAGCUCUUGCAGUAGGUUUUUGCAAGCUAGUGAACGCUG>hsa-mir-1207 MI0006340GCAGGGCUGGCAGGGAGGCUGGGAGGGGCUGGCUGGGUCUGGUAGUGGGCAUCAGCUGGCCCUCAUUUCUUAAGACAGCACUUCUGU>hsa-mir-1208 MI0006341CACCGGCAGAAUCACUGUUCAGACAGGCGGAGACGGGUCUUUCUCGCCCUCUGAUGAGUCACCACUGUGGUGG>hsa-mir-1224 MI0003764GUGAGGACUCGGGAGGUGGAGGGUGGUGCCGCCGGGGCCGGGCGCUGUUUCAGCUCGCUUCUCCCCCCACCUCCUCUCUCCUCAG>hsa-mir-1225 MI0006311GUGGGUACGGCCCAGUGGGGGGGAGAGGGACACGCCCUGGGCUCUGCCCAGGGUGCAGCCGGACUGACUGAGCCCCUGUGCCGCCCCCAG>hsa-mir-1226 MI0006313GUGAGGGCAUGCAGGCCUGGAUGGGGCAGCUGGGAUGGUCCAAAAGGGUGGCCUCACCAGCCCUGUGUUCCCUAG>hsa-mir-1227 MI0006316GUGGGGCCAGGCGGUGGUGGGCACUGCUGGGGUGGGCACAGCAGCCAUGCAGAGCGGGCAUUUGACCCCGUGCCACCCUUUUCCCCAG>hsa-mir-1228 MI0006318GUGGGCGGGGGCAGGUGUGUGGUGGGUGGUGGCCUGCGGUGAGCAGGGCCCUCACACCUGCCUCGCCCCCCAG>hsa-mir-1229 MI0006319GUGGGUAGGGUUUGGGGGAGAGCGUGGGCUGGGGUUCAGGGACACCCUCUCACCACUGCCCUCCCACAG>hsa-mir-1231 MI0006321GUCAGUGUCUGGGCGGACAGCUGCAGGAAAGGGAAGACCAAGGCUUGCUGUCUGUCCAGUCUGCCACCCUACCCUGUCUGUUCUUGCCACAG>hsa-mir-1233-1 MI0006323GUGAGUGGGAGGCCAGGGCACGGCAGGGGGAGCUGCAGGGCUAUGGGAGGGGCCCCAGCGUCUGAGCCCUGUCCUCCCGCAG>hsa-mir-1233-2 MI0015973GUGAGUGGGAGGCCAGGGCACGGCAGGGGGAGCUGCAGGGCUAUGGGAGGGGCCCCAGCGUCUGAGCCCUGUCCUCCCGCAG>hsa-mir-1234 MI0006324GUGAGUGUGGGGUGGCUGGGGGGGGGGGGGGGGGGCCGGGGACGGCUUGGGCCUGCCUAGUCGGCCUGACCACCCACCCCACAG>hsa-mir-1236 MI0006326GUGAGUGACAGGGGAAAUGGGGAUGGACUGGAAGUGGGCAGCAUGGAGCUGACCUUCAUCAUGGCUUGGCCAACAUAAUGCCUCUUCCCCUUGUCUCUCCAG>hsa-mir-1237 MI0006327GUGGGAGGGCCCAGGCGCGGGCAGGGGUGGGGGUGGCAGAGCGCUGUCCCGGGGGCGGGGCCGAAGCGCGGCGACCGUAACUCCUUCUGCUCCGUCCCCCAG>hsa-mir-1238 MI0006328GUGAGUGGGAGCCCCAGUGUGUGGUUGGGGCCAUGGCGGGUGGGCAGCCCAGCCUCUGAGCCUUCCUCGUCUGUCUGCCCCAG>hsa-mir-1243 MI0006373CUAAAACUGGAUCAAUUAUAGGAGUGAAAUAAAGGUCCAUCUCCUGCCUAUUUAUUACUUUGCUUUGGUAAUAAAUCUAUUUUUAAAAGAACC>hsa-mir-1244-1 MI0006379AUCUUAUUCCGAGCAUUCCAGUAACUUUUUUGUGUAUGUACUUAGCUGUACUAUAAGUAGUUGGUUUGUAUGAGAUGGUUAAAAA>hsa-mir-1244-2 MI0015974AUCUUAUUCCGAGCAUUCCAGUAACUUUUUUGUGUAUGUACUUAGCUGUACUAUAAGUAGUUGGUUUGUAUGAGAUGGUUAAAAA>hsa-mir-1244-3 MI0015975AUCUUAUUCCGAGCAUUCCAGUAACUUUUUUGUGUAUGUACUUAGCUGUACUAUAAGUAGUUGGUUUGUAUGAGAUGGUUAAAAA>hsa-mir-1245a MI0006380AUUUAUGUAUAGGCCUUUAGAUCAUCUGAUGUUGAAUACUCUUUAAGUGAUCUAAAGGCCUACAUAUAAA>hsa-mir-1245b MI0017431UUUAUAUGUAGGCCUUUAGAUCACUUAAAGAGUAUUCAACAUCAGAUGAUCUAAAGGCCUAUACAUAAA>hsa-mir-1246 MI0006381UGUAUCCUUGAAUGGAUUUUUGGAGCAGGAGUGGACACCUGACCCAAAGGAAAUCAAUCCAUAGGCUAGCAAU>hsa-mir-1247 MI0006382CCGCUUGCCUCGCCCAGCGCAGCCCCGGCCGCUGGGCGCACCCGUCCCGUUCGUCCCCGGACGUUGCUCUCUACCCCGGGAACGUCGAGACUGGAGCGCCCGAACUGAGCCACCUUCGCGGACCCCGAGAGCGGCG>hsa-mir-1248 MI0006383UUUACCUUCUUGUAUAAGCACUGUGCUAAAAUUGCAGACACUAGGACCAUGUCUUGGUUUUUGCAAUAAUGCUAGCAGAGUACACACAAGAAGAAAAGUAACAGCA>hsa-mir-1249 MI0006384GGGAGGAGGGAGGAGAUGGGCCAAGUUCCCUCUGGCUGGAACGCCCUUCCCCCCCUUCUUCACCUG>hsa-mir-1250 MI0006385CUGUCCCGCUGGCCUGGCAGGUGACGGUGCUGGAUGUGGCCUUUUUGCCUUUUCUAAAGGCCACAUUUUCCAGCCCAUUCAACCUUCCAGAGCCCUCUGAAGUGGCCACAGGC>hsa-mir-1251 MI0006386GUGGACUCUAGCUGCCAAAGGCGCUUCUCCUUCUGAACAGAGCGCUUUGCUCAGCCAGUGUAGACAUGGC>hsa-mir-1252 MI0006434AGAAAGAAGGAAAUUGAAUUCAUUUAGAAAAGAGAAUUCCAAAUGAGCUUAAUUUCCUUUUUUCU>hsa-mir-1253 MI0006387AGCAGCAAGAGAUAGAAUCCAAAAGAGAAGAAGAUCAGCCUGCAGAUGUGGACUGCUAAAUGCAGGCUGAUCUUCUCCCCUUUGGGAUUCUCUUAUGAGAAGCCA>hsa-mir-1254-1 MI0006388GGUGGGAGGAUUGCUUGAGCCUGGAAGCUGGAGCCUGCAGUGAACUAUCAUUGUGCCACUGUACUCCAGCCUAGGCAACAAAAUGAAAUCCUGUCUA>hsa-mir-1254-2 MI0016747CUGAGCCUGGAAGCUGGAGCCUGCAGUGAGCUAUGAUCAUGUCCCUGUACUCUAGCCUGGGCA>hsa-mir-1255a MI0006389AUUGGAAAUCCUUUGAGUUGCUUCUCAAGGAUGAGCAAAGAAAGUAGAUUUUUUAGAUUCUAAAGAAACUAUCUUCUUUGCUCAUCCUUGAGAAGCAACUCCUUAUCCAUUAA>hsa-mir-1255b-1 MI0006435UACGGAUGAGCAAAGAAAGUGGUUUCUUAAAAUGGAAUCUACUCUUUGUGAAGAUGCUGUGAA>hsa-mir-1255b-2 MI0006436UCUUACGGAUGAGCAAAGAAAGUGGUUUGCGCCUCAAGAAACCACUUUCUUUGCUCAUCCAUAAGGA>hsa-mir-1256 MI0006390AGUCAGCCUGUUGAAGCUUUGAAGCUUUGAUGCCAGGCAUUGACUUCUCACUAGCUGUGAAAGUCCUAGCUAAAGAGAAGUCAAUGCAUGACAUCUUGUUUCAAUAGAUGGCUGUUUCA>hsa-mir-1257 MI0006391GCCCUGGGCUUGUGCUUGGGGAGUGAAUGAUGGGUUCUGACCCCCAUGCACCCCUGUGGGCCCCUGGCAUCACUGGCCCCAUCCUUCACCCCUGCCAACCACGCUUGCCCUGUGCCU>hsa-mir-1258 MI0006392CUGUGGCUUCCACGACCUAAUCCUAACUCCUGCGAGUCCCUGGAGUUAGGAUUAGGUCGUGGAAGCCACAGGA>hsa-mir-1260a MI0006394ACCUUUCCAGCUCAUCCCACCUCUGCCACCAAAACACUCAUCGCGGGGUCAGAGGGAGUGCCAAAAAAGGUAA>hsa-mir-1260b MI0014197UCUCCGUUUAUCCCACCACUGCCACCAUUAUUGCUACUGUUCAGCAGGUGCUGCUGGUGGUGAUGGUGAUAGUCUGGUGGGGGCGGUGG>hsa-mir-1261 MI0006396UGCUAUGGAUAAGGCUUUGGCUUAUGGGGAUAUUGUGGUUGAUCUGUUCUAUCCAGAUGACUGAAACUUUCUCCAUAGCAGC>hsa-mir-1262 MI0006397AUCUACAAUGGUGAUGGGUGAAUUUGUAGAAGGAUGAAAGUCAAAGAAUCCUUCUGGGAACUAAUUUUUGGCCUUCAACAAGAAUUGUGAUAU>hsa-mir-1263 MI0006398CUACCCCAAAAUAUGGUACCCUGGCAUACUGAGUAUUUUAAUACUGGCAUACUCAGUAUGCCAUGUUGCCAUAUUUUGGGGUAGCA>hsa-mir-1264 MI0003758AGGUCCUCAAUAAGUAUUUGUUGAAAGAAUAAAUAAACCAACAAGUCUUAUUUGAGCACCUGUUAUGUG>hsa-mir-1265 MI0006401AUGGUUUGGGACUCAGGAUGUGGUCAAGUGUUGUUAAGGCAUGUUCAGGAACAAUACUUGACCACAUUUUGAAUUCCAAACCAUAU>hsa-mir-1266 MI0006403ACAGGUAGUGUCCCUCAGGGCUGUAGAACAGGGCUGGGAUUACUAAAGCCCUGUUCUAUGCCCUGAGGGACACUGAGCAUGUCA>hsa-mir-1267 MI0006404CUCCCAAAUCUCCUGUUGAAGUGUAAUCCCCACCUCCAGCAUUGGGGAUUACAUUUCAACAUGAGAUUUGGAUGAGGA>hsa-mir-1268a MI0006405UAGCCGGGCGUGGUGGUGGGGGCCUGUGGUCCCAGCUACUUUGGAGGCUGAG>hsa-mir-1268b MI0016748ACCCGGGCGUGGUGGUGGGGGUGGGUGCCUGUAAUUCCAGCUAGUUGGGA>hsa-mir-1269a MI0006406UGGAUUGCCUAGACCAGGGAAGCCAGUUGGCAUGGCUCAGUCCAAGUCUGACCACCUGAGGAAUGCCUGGACUGAGCCGUGCUACUGGCUUCCCUGGUCUCCAGC>hsa-mir-1269b MI0016888UGAGGUUUCUGGACUGAGCCAUGCUACUGGCUUCUCUGGUUCUCCAGCUUACAGAUGGCUUAUCAUGGGACCUCU>hsa-mir-1270-1 MI0006407CACAGAGUUAUACUGGAGAUAUGGAAGAGCUGUGUUGGGUAUAAGUAACAGGCUUUUCUUUAUCUUCUAUGUGGCUCUUUGCA>hsa-mir-1270-2 MI0015976CACAGAGUUAUACUGGAGAUAUGGAAGAGCUGUGUUGGGUAUAAGUAACAGGCUUUUCUUUAUCUUCUAUGUGGCUCUUUGCA>hsa-mir-1271 MI0003814CACCCAGAUCAGUGCUUGGCACCUAGCAAGCACUCAGUAAAUAUUUGUUGAGUGCCUGCUAUGUGCCAGGCAUUGUGCUGAGGGCU>hsa-mir-1272 MI0006408CCAGAUCAGAUCUGGGUGCGAUGAUGAUGGCAGCAAAUUCUGAAAACGUGCUCAGUGUCUUUAUAACAGGAAAGCCGUAAACUUAGAAAUGUAGGCUGCAGCUCGUGUGCUCUGUGGUCUGGGCUGGUA>hsa-mir-1273a MI0006409UGAGGCAGGAGAAUUGCUUGAACCCGGGUGGUGGAGGUUGCAGUGAGCCAAGAUUGCGCCACUGCACUCCAGCCUGGGCGACAAAGCAAGACUCUUUCUUGGA>hsa-mir-1273c MI0014171UGCAGCCUGGGCGACAAAACGAGACCCUGUCUUUUUUUUUUUCUGAGACAGAGUCUCGUUCUGUUGCCCAAGCUGGA>hsa-mir-1273d MI0014254GAAUCGCUUGAACCCAUGAGGUUGAGGCUGCAGUGAGCCAAGAUCGUGCCACUGCACUUCAGCCUGGGUGACAAGAGCGAAACUUC>hsa-mir-1273e MI0016059UGAGGCAGGAGAAUUGCUUGAACCCAGGAAGUGGAGGCUGCAGUGAGCCGAGAUCGAGCCACUGUACUCCAGCCUGGGUGACACAGCGAGACUCCAGUCUCA>hsa-mir-1273f MI0018002AGGUGGGAGGAUUGCUUGAGCCUGGGAGAUGGAGGUUGCAGUGAGCUGAGAUCACGCAACUGCACCCCCAGCCUGGGCCAUAGAGUCAGUCCUUGUCUC>hsa-mir-1273g MI0018003GAGGUGGGAGGAUUGCUUGAGUCAGGGUGGUUGAGGCUGCAGUAAGUUGUGAUCAUACCACUGCACUCCAGCCUGAGUGACAGAGCAAGACCUUGUCUCA>hsa-mir-1275 MI0006415CCUCUGUGAGAAAGGGUGUGGGGGAGAGGCUGUCUUGUGUCUGUAAGUAUGCCAAACUUAUUUUCCCCAAGGCAGAGGGA>hsa-mir-1276 MI0006416CCCCAGCUAGGUAAAGAGCCCUGUGGAGACACCUGGAUUCAGAGAACAUGUCUCCACUGAGCACUUGGGCCUUGAUGGCGGCU>hsa-mir-1277 MI0006419ACCUCCCAAAUAUAUAUAUAUAUGUACGUAUGUGUAUAUAAAUGUAUACGUAGAUAUAUAUGUAUUUUUGGUGGGUUU>hsa-mir-1278 MI0006425AUUUGCUCAUAGAUGAUAUGCAUAGUACUCCCAGAACUCAUUAAGUUGGUAGUACUGUGCAUAUCAUCUAUGAGCGAAUAG>hsa-mir-1279 MI0006426AUAUUCACAAAAAUUCAUAUUGCUUCUUUCUAAUGCCAAGAAAGAAGAGUAUAAGAACUUCC>hsa-mir-1280 MI0006437UCUGUCCCACCGCUGCCACCCUCCCCUCUGCCUCAGUGUGCCAGGCAUCAGCACUCACUCACAGAGGCAGGCUGGAUGGCGGGUGGGACAACAG>hsa-mir-1281 MI0006428AGGGGGCACCGGGAGGAGGUGAGUGUCUCUUGUCGCCUCCUCCUCUCCCCCCUU>hsa-mir-1282 MI0006429CCUUCUUCUCGUUUGCCUUUUUCUGCUUCUGCUGCAUGAUCUCCGAGUCCCUGGGGGUAGAGAUGAUGGGGCACUGGGAGGUACCAGAGGGCAAAAAGGAC>hsa-mir-1283-1 MI0003832CUCAAGCUAUGAGUCUACAAAGGAAAGCGCUUUCUGUUGUCAGAAAGAAGAGAAAGCGCUUCCCUUUUGAGGGUUACGGUUUGAGAA>hsa-mir-1283-2 MI0006430CUCAAGCUGUGAGUCUACAAAGGAAAGCGCUUUCUGUUGUCUGAAAGAAAAGAAAUCGCUUCCCUUUGGAGUGUUACGGUUUGAGAA>hsa-mir-1284 MI0006431AUUUUGAUAUAUAAGCCAGUUUAAUGUUUUCUAUACAGACCCUGGCUUUUCUUAAAUUUUAUAUAUUGGAAAGCCCAUGUUUGUAUUGGAAACUGCUGGUUUCUUUCAUACUGAAAAUCU>hsa-mir-1285-1 MI0006346UGUAGAGAUAGGAUCUCACUUUGUUGCCCAGGCUGGUCUCAAACUCCUGGUCUGGGCAACAAAGUGAGACCUUAUCUCUACAAG>hsa-mir-1285-2 M
I0006347UUUGGGAGGCCGAGGCUGGUGCAUCACUUGAGCCCAGCAAUUUGAGACCAAUCUGGGCAACAAAGUGAGACCUCCGUCUCUACAAAGA>hsa-mir-1286 MI0006348UGUCCUCUGGGGACUCAGCUUGCUCUGGCUGCUGGAUUGAAUUAGCUGCAGGACCAAGAUGAGCCCUUGGUGGAGACA>hsa-mir-1287 MI0006349GUUGUGCUGUCCAGGUGCUGGAUCAGUGGUUCGAGUCUGAGCCUUUAAAAGCCACUCUAGCCACAGAUGCAGUGAUUGGAGCCAUGACAA>hsa-mir-1288 MI0006432GAGGGUGUUGAUCAGCAGAUCAGGACUGUAACUCACCAUAGUGGUGGACUGCCCUGAUCUGGAGACCACUGCCUU>hsa-mir-1289-1 MI0006350UUCUCAAUUUUUAGUAGGAAUUAAAAACAAAACUGGUAAAUGCAGACUCUUGGUUUCCACCCCCAGAGAAUCCCUAAACCGGGGGUGGAGUCCAGGAAUCUGCAUUUUAGAAAGUACCCAGGGUGAUUCUGAUAAUUGGGAACA>hsa-mir-1289-2 MI0006351CCACGGUCCUAGUUAAAAAGGCACAUUCCUAGACCCUGCCUCAGAACUACUGAACAGAGUCACUGGGUGUGGAGUCCAGGAAUCUGCAUUUUUACCCCUAUCGCCCCCGCC>hsa-mir-1290 MI0006352GAGCGUCACGUUGACACUCAAAAAGUUUCAGAUUUUGGAACAUUUCGGAUUUUGGAUUUUUGGAUCAGGGAUGCUCAA>hsa-mir-1291 MI0006353GGUAGAAUUCCAGUGGCCCUGACUGAAGACCAGCAGUUGUACUGUGGCUGUUGGUUUCAAGCAGAGGCCUAAAGGACUGUCUUCCUG>hsa-mir-1292 MI0006433CCUGGGAACGGGUUCCGGCAGACGCUGAGGUUGCGUUGACGCUCGCGCCCCGGCUCCCGUUCCAGG>hsa-mir-1293 MI0006355AGGUUGUUCUGGGUGGUCUGGAGAUUUGUGCAGCUUGUACCUGCACAAAUCUCCGGACCACUUAGUCUUUA>hsa-mir-1294 MI0006356CACCUAAUGUGUGCCAAGAUCUGUUCAUUUAUGAUCUCACCGAGUCCUGUGAGGUUGGCAUUGUUGUCUGGCAUUGUCUGAUAUACAACAGUGCCAACCUCACAGGACUCAGUGAGGUGAAACUGAGGAUUAGGAAGGUGUA>hsa-mir-1295a MI0006357AGGACAUUUUGCCCAGAUCCGUGGCCUAUUCAGAAAUGUGGCCUGUGAUUAGGCCGCAGAUCUGGGUGAAAUGUCCUCC>hsa-mir-1295b MI0019146CACCCAGAUCUGCGGCCUAAUCACAGGCCACAUUUCUGAAUAGGCCACGGAUCUGGGCAA>hsa-mir-1296 MI0003780ACCUACCUAACUGGGUUAGGGCCCUGGCUCCAUCUCCUUUAGGAAAACCUUCUGUGGGGAGUGGGGCUUCGACCCUAACCCAGGUGGGCUGU>hsa-mir-1297 MI0006358UGUUUAUCUCUAGGGUUGAUCUAUUAGAAUUACUUAUCUGAGCCAAAGUAAUUCAAGUAAUUCAGGUGUAGUGAAAC>hsa-mir-1298 MI0003938AGACGAGGAGUUAAGAGUUCAUUCGGCUGUCCAGAUGUAUCCAAGUACCCUGUGUUAUUUGGCAAUAAAUACAUCUGGGCAACUGACUGAACUUUUCACUUUUCAUGACUCA>hsa-mir-1299 MI0006359CCUCAUGGCAGUGUUCUGGAAUCCUACGUGAGGGACAAUCAUUCAGACCCACGUAGCAGUGUUCUGGAAUUCUGUGUGAGGGA>hsa-mir-1301 MI0003815GGAUUGUGGGGGGUCGCUCUAGGCACCGCAGCACUGUGCUGGGGAUGUUGCAGCUGCCUGGGAGUGACUUCACACAGUCCUC>hsa-mir-1302-1 MI0006362CAGAAAGCCCAGUUAAAUUUGAAUUUCAAGUAAACAAUGAAUAAUUGUGUAUGUAAGAAUAUCCCAUACAAUAUUUGGGACAUACUUAUGCUAAAAAUUAUUCCUUGCUUAUCUGAAAUUCAAAUGUAACUAGGAUUCCUGUA>hsa-mir-1302-10 MI0015979GGAUGCCCAGCUAGUUUGAAUUUUAGAUAAACAACGAAUAAUUUCGUAGCAUAAAUAUGUCCCAAGCUUAGUUUGGGACAUACUUAUGCUAAAAAACAUUAUUGGUUGUUUAUCUGAGAUUCAGAAUUAAGCAUUUUA>hsa-mir-1302-11 MI0015980GGAUGCCCAGCUAGUUUGAAUUUUAGAUAAACAACGAAUAAUUUCGUAGCAUAAAUAUGUCCCAAGCUUAGUUUGGGACAUACUUAUGCUAAAAAACAUUAUUGGUUGUUUAUCUGAGAUUCAGAAUUAAGCAUUUUA>hsa-mir-1302-2 MI0006363GGAUGCCCAGCUAGUUUGAAUUUUAGAUAAACAACGAAUAAUUUCGUAGCAUAAAUAUGUCCCAAGCUUAGUUUGGGACAUACUUAUGCUAAAAAACAUUAUUGGUUGUUUAUCUGAGAUUCAGAAUUAAGCAUUUUA>hsa-mir-1302-3 MI0006364GGAUGCCCAGCUAGUUUGAAUUUUAGAUAAACAACGAAUAAUUUCGUAGCAUAAAUAUUUCCCAAGCUUAGUUUGGGACAUACUUAUGCUAAAAAACAUUAUUGGUUGUUUAUCUGAGAUUCAAAAUUAAGCAUUUUA>hsa-mir-1302-4 MI0006365AAUGCAGAAGCACAGCUAAAAUUUGAAUUUCAGAUAAACAAAUUUUUCUUAGAAUAAGUAUGUCUCCAUGCAACAUUUGGGACAUACUUAUGCUAAAAUAUUAUUUGUGUUUCAUCUGAAAUUCAAAUUCAACUGGACAUCCUGUAUUUU>hsa-mir-1302-5 MI0006366UGCCCGGCCUCCCAUUAAAUUGGUUUUUCAGACAAAUCACAAAUUUGUUUAGGUAUAAGUAUAUCCCAUGUAAUCUUUGGGACAUACUUAUGCUAAAAUAAUUGUUCCUUGUUGAUUGGAAAUUUUAAUUUUAAUUAGGUGUCCUGUAUU>hsa-mir-1302-6 MI0006367AACAAAUAAUUUGGUAAUAUAUGUAUGGCCCACACAAUAUUUAGGACAACAAUAUUUGGGACAUACUUAUGCUAAAAAAGUAUUUGUUGA>hsa-mir-1302-7 MI0006368ACAACAUGUUUUUAGGACAUGUAUGUCUGGUGCAAUAAUUGGGACAUACUUAUGCUAAAAAAAUUAGUGUUC>hsa-mir-1302-8 MI0006369CCCAUUUAAACUUGAAUUUCAUAUAAACACCGUAAUUUUCAGCAUUAGUGUAUCACAUGCAGUAUUUGGGACAUACUUAUGCUAAAAAAUUAGGUGGUGUUGAUCUGAAAUUCCAGUGUAGAUGGGCA>hsa-mir-1302-9 MI0015978GGAUGCCCAGCUAGUUUGAAUUUUAGAUAAACAACGAAUAAUUUCGUAGCAUAAAUAUGUCCCAAGCUUAGUUUGGGACAUACUUAUGCUAAAAAACAUUAUUGGUUGUUUAUCUGAGAUUCAGAAUUAAGCAUUUUA>hsa-mir-1303 MI0006370GGCUGGGCAACAUAGCGAGACCUCAACUCUACAAUUUUUUUUUUUUUAAAUUUUAGAGACGGGGUCUUGCUCUGUUGCCAGGCUUU>hsa-mir-1304 MI0006371AAACACUUGAGCCCAGCGGUUUGAGGCUACAGUGAGAUGUGAUCCUGCCACAUCUCACUGUAGCCUCGAACCCCUGGGCUCAAGUGAUUCA>hsa-mir-1305 MI0006372AAGAUCCUGCUGUUUCUACCAUUAGUUUUGAAUGUUUAUUGUAAAGAUACUUUUCAACUCUAAUGGGAGAGACAGCAGGAUUCUCC>hsa-mir-1306 MI0006443GUGAGCAGUCUCCACCACCUCCCCUGCAAACGUCCAGUGGUGCAGAGGUAAUGGACGUUGGCUCUGGUGGUGAUGGACAGUCCGA>hsa-mir-1307 MI0006444CAUCAAGACCCAGCUGAGUCACUGUCACUGCCUACCAAUCUCGACCGGACCUCGACCGGCUCGUCUGUGUUGCCAAUCGACUCGGCGUGGCGUCGGUCGUGGUAGAUAGGCGGUCAUGCAUACGAAUUUUCAGCUCUUGUUCUGGUGAC>hsa-mir-1321 MI0006652ACAUUAUGAAGCAAGUAUUAUUAUCCCUGUUUUACAAAUAAGGAAAUAAACUCAGGGAGGUGAAUGUGAUCAAAGAUAG>hsa-mir-1322 MI0006653AGUAUCAUGAAUUAGAAACCUACUUAUUACAUAGUUUACAUAAGAAGCGUGAUGAUGCUGCUGAUGCUGUA>hsa-mir-1323 MI0003786ACUGAGGUCCUCAAAACUGAGGGGCAUUUUCUGUGGUUUGAAAGGAAAGUGCACCCAGUUUUGGGGAUGUCAA>hsa-mir-1324 MI0006657CCUGAAGAGGUGCAUGAAGCCUGGUCCUGCCCUCACUGGGAACCCCCUUCCCUCUGGGUACCAGACAGAAUUCUAUGCACUUUCCUGGAGGCUCCA>hsa-mir-1343 MI0017320GCUGGCGUCGGUGCUGGGGAGCGGCCCCCGGGUGGGCCUCUGCUCUGGCCCCUCCUGGGGCCCGCACUCUCGCUCUGGGCCCGC>hsa-mir-1468 MI0003782GGUGGGUGGUUUCUCCGUUUGCCUGUUUCGCUGAUGUGCAUUCAACUCAUUCUCAGCAAAAUAAGCAAAUGGAAAAUUCGUCCAUC>hsa-mir-1469 MI0007074CUCGGCGCGGGGCGCGGGCUCCGGGUUGGGGCGAGCCAACGCCGGGG>hsa-mir-1470 MI0007075GCCCUCCGCCCGUGCACCCCGGGGCAGGAGACCCCGCGGGACGCGCCGAGGUAGGGGGGAC>hsa-mir-1471 MI0007076GCCCGCGUGUGGAGCCAGGUGUAGAGGCGGAGCACAGCUGGCUCUAAUUUGAGGGGC>hsa-mir-1537 MI0007258ACAGCUGUAAUUAGUCAGUUUUCUGUCCUGUCCACACAGAAAACCGUCUAGUUACAGUUGU>hsa-mir-1538 MI0007259GGGAACAGCAGCAACAUGGGCCUCGCUUCCUGCCGGCGCGGCCCGGGCUGCUGCUGUUCCU>hsa-mir-1539 MI0007260GGCUCUGCGGCCUGCAGGUAGCGCGAAAGUCCUGCGCGUCCCAGAUGCCC>hsa-mir-1587 MI0016905UUUGGGCUGGGCUGGGUUGGGCAGUUCUUCUGCUGGACUCACCUGUGACCAGC>hsa-mir-1825 MI0008193AGAGACUGGGGUGCUGGGCUCCCCUAGACUAGGACUCCAGUGCCCUCCUCUCC>hsa-mir-1827 MI0008195UCAGCAGCACAGCCUUCAGCCUAAAGCAAUGAGAAGCCUCUGAAAGGCUGAGGCAGUAGAUUGAAU>hsa-mir-1908 MI0008329CGGGAAUGCCGCGGCGGGGACGGCGAUUGGUCCGUAUGUGUGGUGCCACCGGCCGCCGGCUCCGCCCCGGCCCCCGCCCC>hsa-mir-1909 MI0008330CAUCCAGGACAAUGGUGAGUGCCGGUGCCUGCCCUGGGGCCGUCCCUGCGCAGGGGCCGGGUGCUCACCGCAUCUGCCCC>hsa-mir-1910 MI0008331UGUCCCUUCAGCCAGUCCUGUGCCUGCCGCCUUUGUGCUGUCCUUGGAGGGAGGCAGAAGCAGGAUGACAAUGAGGGCAA>hsa-mir-1911 MI0008332UCGGCAUCUGCUGAGUACCGCCAUGUCUGUUGGGCAUCCACAGUCUCCCACCAGGCAUUGUGGUCUCCGCUGACGCUUUG>hsa-mir-1912 MI0008333CUCUAGGAUGUGCUCAUUGCAUGGGCUGUGUAUAGUAUUAUUCAAUACCCAGAGCAUGCAGUGUGAACAUAAUAGAGAUU>hsa-mir-1913 MI0008334ACCUCUACCUCCCGGCAGAGGAGGCUGCAGAGGCUGGCUUUCCAAAACUCUGCCCCCUCCGCUGCUGCCAAGUGGCUGGU>hsa-mir-1914 MI0008335CGUGUGAGCCCGCCCUGUGCCCGGCCCACUUCUGCUUCCUCUUAGCGCAGGAGGGGUCCCGCACUGGGAGGGGCCCUCAC>hsa-mir-1915 MI0008336UGAGAGGCCGCACCUUGCCUUGCUGCCCGGGCCGUGCACCCGUGGGCCCCAGGGCGACGCGGCGGGGGCGGCCCUAGCGA>hsa-mir-1972-1 MI0009982UAUAGGCAUGUGCCACCACACCUGGCUUAAAUGUGUCAUUUAAAAAUUCAGGCCAGGCACAGUGGCUCAUGCCUGUA>hsa-mir-1972-2 MI0015977UAUAGGCAUGUGCCACCACACCUGGCUUAAAUGUGUCAUUUAAAAAUUCAGGCCAGGCACAGUGGCUCAUGCCUGUA>hsa-mir-1973 MI0009983UAUGUUCAACGGCCAUGGUAUCCUGACCGUGCAAAGGUAGCAUA>hsa-mir-1976 MI0009986GCAGCAAGGAAGGCAGGGGUCCUAAGGUGUGUCCUCCUGCCCUCCUUGCUGU>hsa-mir-2052 MI0010486CUGUUUUGAUAACAGUAAUGUCCCUUUAGUUCAAAGUUACCAGCUAUCAAAACAA>hsa-mir-2053 MI0010487CUUGCCAUGUAAAUACAGAUUUAAUUAACAUUUGCAACCUGUGAAGAUGCAAAACUUUAAGUGUUAAUUAAACCUCUAUUUACAUAGCAAG>hsa-mir-2054 MI0010488CUGUAAUAUAAAUUUAAUUUAUUCUCUAUCAUUAAAAAAUGUAUUACAG>hsa-mir-2110 MI0010629CAGGGGUUUGGGGAAACGGCCGCUGAGUGAGGCGUCGGCUGUGUUUCUCACCGCGGUCUUUUCCUCCCACUCUUG>hsa-mir-2113 MI0003939UUUUCAAAGCAAUGUGUGACAGGUACAGGGACAAAUCCCGUUAAUAAGUAAGAGGAUUUGUGCUUGGCUCUGUCACAUGCCACUUUGAAAA>hsa-mir-2114 MI0010633CCUCCAUGCUCCUAGUCCCUUCCUUGAAGCGGUCGGAUAAUCACAUGACGAGCCUCAAGCAAGGGACUUCAAGCUGGUGG>hsa-mir-2115 MI0010634ACUGUCAUCCCACUGCUUCCAGCUUCCAUGACUCCUGAUGGAGGAAUCACAUGAAUUCAUCAGAAUUCAUGGAGGCUAGAAGCAGUAUGAGGAUCAUUUA>hsa-mir-2116 MI0010635GACCUAGGCUAGGGGUUCUUAGCAUAGGAGGUCUUCCCAUGCUAAGAAGUCCUCCCAUGCCAAGAACUCCCAGACUAGGA>hsa-mir-2117 MI0010636GCUCUGAUUUACUUCUGUCCGGCAUGGUGAACAGCAGGAUUGGCUGUAGCUGUUCUCUUUGCCAAGGACAGAUCUGAUCU>hsa-mir-2276 MI0011282GUGUUCUUCCAGUCCGCCCUCUGUCACCUUGCAGACGGCUUUCUCUCCGAAUGUCUGCAAGUGUCAGAGGCGAGGAGUGGCAGCUGCAU>hsa-mir-2277 MI0011284GUGCUUCCUGCGGGCUGAGCGCGGGCUGAGCGCUGCCAGUCAGCGCUCACAUUAAGGCUGACAGCGCCCUGCCUGGCUCGGCCGGCGAAGCUC>hsa-mir-2278 MI0011285GUGCUGCAGGUGUUGGAGAGCAGUGUGUGUUGCCUGGGGACUGUGUGGACUGGUAUCACCCAGACAGCUUGCACUGACUCCAGACCCUGCCGUCAU>hsa-mir-2355 MI0015873CAGACGUGUCAUCCCCAGAUACAAUGGACAAUAUGCUAUUAUAAUCGUAUGGCAUUGUCCUUGCUGUUUGGAGAUAAUACUGCUGAC>hsa-mir-2392 MI0016870AUGGUCCCUCCCAAUCCAGCCAUUCCUCAGACCAGGUGGCUCCCGAGCCACCCCAGGCUGUAGGAUGGGGGUGAGAGGUGCUAG>hsa-mir-2467 MI0017432GGACAGGCACCUGAGGCUCUGUUAGCCUUGGCUCUGGGUCCUGCUCCUUAGAGCAGAGGCAGAGAGGCUCAGGGUCUGUCU>hsa-mir-2681 MI0012062GCCCCCUUUUCACGCAUUUGUGUUUUACCACCUCCAGGAGACUGCCCAAAGACUCUUCAGUAUCAUGGAGUUGGUAAAGCACAGAUGCAUGAAUAAUUCAACGUG>hsa-mir-2682 MI0012063ACCUUCCUGAAAGAGGUUGGGGCAGGCAGUGACUGUUCAGACGUCCAAUCUCUUUGGGACGCCUCUUCAGCGCUGUCUUCCCUGCCUCUGCCUUUAGGACGAGUCUCAAA>hsa-mir-2861 MI0013006GGCGCCUCUGCAGCUCCGGCUCCCCCUGGCCUCUCGGGAACUACAAGUCCCAGGGGGCCUGGCGGUGGGCGGCGGGCGGAAGAGGCGGGG>hsa-mir-2909 MI0013083GGUGUUAGGGCCAACAUCUCUUGGUCUUUCCCCUGUGGUCCCAAGAUGGCUGUUGCAACUUAACGCCAU>hsa-mir-2964a MI0017299GGAGCUCAGCCACAGAUGUCCAGCCACAAUUCUCGGUUGGCCGCAGACUCGUACAAGAAUUGCGUUUGGACAAUCAGUGGCGAAGCCC>hsa-mir-3064 MI0017375GGUCUGGCUGUUGUGGUGUGCAAAACUCCGUACAUUGCUAUUUUGCCACACUGCAACACCUUACAG>hsa-mir-3065 MI0014228CUGCCCUCUUCAACAAAAUCACUGAUGCUGGAGUCGCCUGAGUCAUCACUCAGCACCAGGAUAUUGUUGGAGAGGACAG>hsa-mir-3074 MI0014181GCUCGACUCCUGUUCCUGCUGAACUGAGCCAGUGUGUAAAAUGAGAACUGAUAUCAGCUCAGUAGGCACCGGAGGGCGGGU>hsa-mir-3115 MI0014127UCUGAAUAUGGGUUUACUAGUUGGUGGUGAAUUCAUGAGUCGCCAACUAUUAGGCCUUUAUGUCCAGA>hsa-mir-3116-1 MI0014128CUUUAUUGAGUCCCUACUAUGUUCCAGGCACUGGGUAUCGUAGGUGCCUGGAACAUAGUAGGGACUCAAUAAAG>hsa-mir-3116-2 MI0014129UAUUGAGUCCCUACUAUGUUCCAGGCACCUACGAUACCCAGUGCCUGGAACAUAGUAGGGACUCAAUA>hsa-mir-3117 MI0014130CCCUAAAGGGCCAGACACUAUACGAGUCAUAUAAGGGAAGGCAUUAUAGGACUCAUAUAGUGCCAGGUGUUUUGUGGG>hsa-mir-3118-1 MI0014131CACACUACAAUAAUUUUCAUAAUGCAAUCACACAUAAUCACUAUGUGACUGCAUUAUGAAAAUUCUUGUAGUGUG>hsa-mir-3118-2 MI0014132ACACUACAAUAAUUUUCAUAAUGCAAUCACACAUAAUCACUAUGUGACUGCAUUAUGAAAAUUCUUGUAGUGU>hsa-mir-3118-3 MI0014133CACACUACAAUAAUUUUCAUAAUGCAAUCACACAUAAUCACUAUGUGACUGCAUUAUGAAAAUUCUUGUA
GUGUG>hsa-mir-3118-4 MI0014207CAUACUACAAUAAUUUUCAUAAUGCAAUCACACACAAUCACCGUGUGACUGCAUUAUGAAAAUUCUUCUAGUGUG>hsa-mir-3118-5 MI0014243CACACAUACAAUAAUAUUCAUAAUGCAAUCACACACAAUCACCAUGUGACUGCAUUAUGAAAAUUCUUCUAGUGUG>hsa-mir-3118-6 MI0015981CAUACUACAAUAAUUUUCAUAAUGCAAUCACACACAAUCACCGUGUGACUGCAUUAUGAAAAUUCUUCUAGUGUG>hsa-mir-3119-1 MI0014134AUUAACUCUGGCUUUUAACUUUGAUGGCAAAGGGGUAGCUAAACAAUCUAUGUCUUUGCCAUCAAAGUUAAAAGCCAUAGUUAAU>hsa-mir-3119-2 MI0014135AUUAACUAUGGCUUUUAACUUUGAUGGCAAAGACAUAGAUUGUUUAGCUACCCCUUUGCCAUCAAAGUUAAAAGCCAGAGUUAAU>hsa-mir-3120 MI0014136GUCAUGUGACUGCCUGUCUGUGCCUGCUGUACAGGUGAGCGGAUGUUCUGCACAGCAAGUGUAGACAGGCAGACACAUGAC>hsa-mir-3121 MI0014137AAAUGGUUAUGUCCUUUGCCUAUUCUAUUUAAGACACCCUGUACCUUAAAUAGAGUAGGCAAAGGACAGAAACAUUU>hsa-mir-3122 MI0014138ACCAGCUCUGUUGGGACAAGAGGACGGUCUUCUUUUGGAAGGAAGACCAUCAUCUUGUCCGAAGAGAGCUGGU>hsa-mir-3123 MI0014139AUGGAUUUGAUUGAAUGAUUCUCCCAUUUCCACAUGGAGAGUGGAGCCCAGAGAAUUGUUUAAUCAUGUAUCCAU>hsa-mir-3124 MI0014140GCGGGCUUCGCGGGCGAAGGCAAAGUCGAUUUCCAAAAGUGACUUUCCUCACUCCCGUGAAGUCGGC>hsa-mir-3125 MI0014142GAGAAUGGGUAGAGGAAGCUGUGGAGAGAACUCACGGUGCCUGUGGUUCGAGAUCCCCGCCUUCCUCCUCCUUUCCUC>hsa-mir-3126 MI0014143AUGAUUAUAUGAGGGACAGAUGCCAGAAGCACUGGUUAUGAUUUGCAUCUGGCAUCCGUCACACAGAUAAUUAU>hsa-mir-3127 MI0014144GGCCAGGCCCAUCAGGGCUUGUGGAAUGGGAAGGAGAAGGGACGCUUCCCCUUCUGCAGGCCUGCUGGGUGUGGCU>hsa-mir-3128 MI0014145UUCCUCUGGCAAGUAAAAAACUCUCAUUUUCCUUAAAAAAUGAGAGUUUUUUACUUGCAAUAGGAA>hsa-mir-3129 MI0014146GUACUUGGGCAGUAGUGUAGAGAUUGGUUUGCCUGUUAAUGAAUUCAAACUAAUCUCUACACUGCUGCCCAAGAGC>hsa-mir-3130-1 MI0014147CUUGUCAUGUCUUACCCAGUCUCCGGUGCAGCCUGUUGUCAAGGCUGCACCGGAGACUGGGUAAGACAUGACAAG>hsa-mir-3130-2 MI0014148CUUGUCAUGUCUUACCCAGUCUCCGGUGCAGCCUUGACAACAGGCUGCACCGGAGACUGGGUAAGACAUGACAAG>hsa-mir-3131 MI0014151GAGUCGAGGACUGGUGGAAGGGCCUUUCCCCUCAGACCAAGGCCCUGGCCCCAGCUUCUUCUC>hsa-mir-3132 MI0014152GGUGGGAUGGGUAGAGAAGGAGCUCAGAGGACGGUGCGCCUUGUUUCCCUUGAGCCCUCCCUCUCUCAUCCCACC>hsa-mir-3133 MI0014153CAGAAAUUGUAAAGAACUCUUAAAACCCAAUAGUAAAAAGACAACCUGUUGAGUUUUAAGAGUUCUUUAUAUAUUCUG>hsa-mir-3134 MI0014155UGUAUCCAAUGUGUAGUCUUUUAUCCCUCACAUGGAGUAAAAUAUGAUGGAUAAAAGACUACAUAUUGGGUACA>hsa-mir-3135a MI0014156UCACUUUGGUGCCUAGGCUGAGACUGCAGUGGUGCAAUCUCAGUUCACUGCAGCCUUGACCUCCUGGGCUCAGGUGA>hsa-mir-3135b MI0016809UGCCCAGGCUGGAGCGAGUGCAGUGGUGCAGUCAGUCCUAGCUCACUGCAGCCUCGAACUCCUGGGCU>hsa-mir-3136 MI0014158AAUAUGAAACUGACUGAAUAGGUAGGGUCAUUUUUCUGUGACUGCACAUGGCCCAACCUAUUCAGUUAGUUCCAUAUU>hsa-mir-3137 MI0014160UACAGGUCUGUAGCCUGGGAGCAAUGGGGUGUAUGGUAUAGGGGUAGCCUCGUGCUCCUGGGCUACAAACCUGUA>hsa-mir-3138 MI0014161CCCUCCUCGGCACUUCCCCCACCUCACUGCCCGGGUGCCCACAAGACUGUGGACAGUGAGGUAGAGGGAGUGCCGAGGAGGG>hsa-mir-3139 MI0014162GGCUCAGAGUAGGAGCUCAACAGAUGCCUGUUGACUGAAUAAUAAACAGGUAUCGCAGGAGCUUUUGUUAUGUGCC>hsa-mir-3140 MI0014163CCUCUUGAGGUACCUGAAUUACCAAAAGCUUUAUGUAUUCUGAAGUUAUUGAAAAUAAGAGCUUUUGGGAAUUCAGGUAGUUCAGGAGUG>hsa-mir-3141 MI0014165UCACCCGGUGAGGGCGGGUGGAGGAGGAGGGUCCCCACCAUCAGCCUUCACUGGGACGGGA>hsa-mir-3142 MI0014166UUCAGAAAGGCCUUUCUGAACCUUCAGAAAGGCUGCUGAAUCUUCAGAAAGGCCUUUCUGAACCUUCAGAAAGGCUGCUGAA>hsa-mir-3143 MI0014167UAGAUAACAUUGUAAAGCGCUUCUUUCGCGGUUGGGCUGGAGCAACUCUUUACAAUGUUUCUA>hsa-mir-3144 MI0014169AACUACACUUUAAGGGGACCAAAGAGAUAUAUAGAUAUCAGCUACCUAUAUACCUGUUCGGUCUCUUUAAAGUGUAGUU>hsa-mir-3145 MI0014170UAUAUGAGUUCAACUCCAAACACUCAAAACUCAUUGUUGAAUGGAAUGAGAUAUUUUGAGUGUUUGGAAUUGAACUCGUAUA>hsa-mir-3146 MI0014172GCUAAGUCCCUUCUUUCUAUCCUAGUAUAACUUGAAGAAUUCAAAUAGUCAUGCUAGGAUAGAAAGAAUGGGACUUGGC>hsa-mir-3147 MI0014173GUCCGGGUUGGGCAGUGAGGAGGGUGUGACGCCGCGAAGUGCACCUCGCCCUUGUCCAACUCGGAC>hsa-mir-3148 MI0014175GAGUUAAGAUGGAAAAAACUGGUGUGUGCUUAUUGAUGUAGCCAACAAGCAUACAUCAGUUUUUUCCAACUUAACUC>hsa-mir-3149 MI0014176AUACAUACAUGUACACACACAUGUCAUCCACACACAUACAUAUAUAUAUGUUUGUAUGGAUAUGUGUGUGUAUGUGUGUGUAU>hsa-mir-3150a MI0014177GGGAAGCAGGCCAACCUCGACGAUCUCCUCAGCACCUGAACGCCAAGGCUGGGGAGAUCCUCGAGGUUGGCCUGCUUUCC>hsa-mir-3150b MI0016426GAGGGAAAGCAGGCCAACCUCGAGGAUCUCCCCAGCCUUGGCGUUCAGGUGCUGAGGAGAUCGUCGAGGUUGGCCUGCUUCCCCUC>hsa-mir-3151 MI0014178GGGGUGAUGGGUGGGGCAAUGGGAUCAGGUGCCUCAAAGGGCAUCCCACCUGAUCCCACAGCCCACCUGUCACCCC>hsa-mir-3152 MI0014179GUGCAGAGUUAUUGCCUCUGUUCUAACACAAGACUAGGCUUCCCUGUGUUAGAAUAGGGGCAAUAACUCUGCAC>hsa-mir-3153 MI0014180GACAAAUUUUAAAUGUCCCUGUCCCCUUCCCCCCAAUUAAAGUAGAUUGGGGGAAAGCGAGUAGGGACAUUUAAAAUUUGUU>hsa-mir-3154 MI0014182GGCCCCUCCUUCUCAGCCCCAGCUCCCGCUCACCCCUGCCACGUCAAAGGAGGCAGAAGGGGAGUUGGGAGCAGAGAGGGGACC>hsa-mir-3155a MI0014183UCCGGGCAUCACCUCCCACUGCAGAGCCUGGGGAGCCGGACAGCUCCCUUCCCAGGCUCUGCAGUGGGAACUGAUGCCUGGA>hsa-mir-3155b MI0016839CCACUGCAGAGCCUGGGAAGGGAGCUGUCCGGCUCCCCAGGCUCUGCAGUGGGAGU>hsa-mir-3156-1 MI0014184GCAGAAGAAAGAUCUGGAAGUGGGAGACACUUUUACUAUAUAUAGUGGCUCCCACUUCCAGAUCUUUCUCUCUGU>hsa-mir-3156-2 MI0014230UGCAGAAGAAAGAUCUGGAAGUGGGAGACACUUUCACUAUAUAUAGUGGCUCCCACUUCCAGAUCUUUCUCUCUGUA>hsa-mir-3156-3 MI0014242UGCAGAAGAAAGAUCUGGAAGUGGGAGACACUUUCACUAUAUAUAGUGGCUCCCACUUCCUGAUCUUUCUCUCUGUA>hsa-mir-3157 MI0014185GGGAAGGGCUUCAGCCAGGCUAGUGCAGUCUGCUUUGUGCCAACACUGGGGUGAUGACUGCCCUAGUCUAGCUGAAGCUUUUCCC>hsa-mir-3158-1 MI0014186AUUCAGGCCGGUCCUGCAGAGAGGAAGCCCUUCUGCUUACAGGUAUUGGAAGGGCUUCCUCUCUGCAGGACCGGCCUGAAU>hsa-mir-3158-2 MI0014187AUUCAGGCCGGUCCUGCAGAGAGGAAGCCCUUCCAAUACCUGUAAGCAGAAGGGCUUCCUCUCUGCAGGACCGGCCUGAAU>hsa-mir-3159 MI0014188CCAAAGUCCUAGGAUUACAAGUGUCGGCCACGGGCUGGGCACAGUGGCUCACGCCUGUAAUCCCAGCAUUUUGG>hsa-mir-3160-1 MI0014189GGACCUGCCCUGGGCUUUCUAGUCUCAGCUCUCCUCCAGCUCAGCUGGUCAGGAGAGCUGAGACUAGAAAGCCCAGGGCAGGUUC>hsa-mir-3160-2 MI0014190ACCUGCCCUGGGCUUUCUAGUCUCAGCUCUCCUGACCAGCUGAGCUGGAGGAGAGCUGAGACUAGAAAGCCCAGGGCAGGU>hsa-mir-3161 MI0014191CCUCGAGAGCUGAUAAGAACAGAGGCCCAGAUUGAAGUUGAAUAGUGCUGGGCCUUUGUUUUUACCAAGUUCCCUGG>hsa-mir-3162 MI0014192CUGACUUUUUUAGGGAGUAGAAGGGUGGGGAGCAUGAACAAUGUUUCUCACUCCCUACCCCUCCACUCCCCAAAAAAGUCAG>hsa-mir-3163 MI0014193UUCCUCAUCUAUAAAAUGAGGGCAGUAAGACCUUCCUUCCUUGUCUUACUACCCCCAUUUUAUAGAUGAGGAA>hsa-mir-3164 MI0014194CUUGGAAACUGUGACUUUAAGGGAAAUGGCGCACAGCAGACCCUGCAAUCAUGCCGUUUUGCUUGAAGUCGCAGUUUCCCAGG>hsa-mir-3165 MI0014195CCGGUGGCAAGGUGGAUGCAAUGUGACCUCAACUCUUGGUCCUCUGAGGUCACAUUGUAUCCACCUUACCACUGG>hsa-mir-3166 MI0014196AAAUUUUUUUGAGGCCAGUAGGCAUUGUCUGCGUUAGGAUUUCUGUAUCAUCCUCCUAACGCAGACAAUGCCUACUGGCCUAAGAAAAAUUU>hsa-mir-3167 MI0014198GGCUGUGGAGGCACCAGUAUUUCUGAAAUUCUUUUUUCUGAAAUUCUUCAGGAAGGAUUUCAGAAAUACUGGUGUCCCGACAGCC>hsa-mir-3168 MI0014199AAGAUCAUGAGUUCUACAGUCAGACAGCCUGAGUUGGAGGCUCAUCUUCACUUCUUGCUGUGUGACCCUGGGCCAGUGACUU>hsa-mir-3169 MI0014200AUGUGAAAACAUAGGACUGUGCUUGGCACAUAGCACAAAGUCUUAUGGUACUGUGUGCCAAGCAUAGUCCUGUGUUUUUACAU>hsa-mir-3170 MI0014201CUGGUAACACUGGGGUUCUGAGACAGACAGUGUUAGCUCCAGAAGCAUUGCCUGUCUUAGAACCCCUAUGUUACCAG>hsa-mir-3171 MI0014202UAUAUAUAGAGAUGUAUGGAAUCUGUAUAUAUCUAUAUAUAUGUGUAUAUAUAGAUUCCAUAAAUCUAUAUAUG>hsa-mir-3173 MI0014204UCCCUGCCCUGCCUGUUUUCUCCUUUGUGAUUUUAUGAGAACAAAGGAGGAAAUAGGCAGGCCAGGGA>hsa-mir-3174 MI0014208GUUACCUGGUAGUGAGUUAGAGAUGCAGAGCCCUGGGCUCCUCAGCAAACCUACUGGAUCUGCAUUUUAAUUCACAUGCAUGGUAAU>hsa-mir-3175 MI0014209CCUGGGGGGCGGGGAGAGAACGCAGUGACGUCUGGCCGCGUGCGCAUGUCGGGCGCUUUCUCCUCCCCCUACCCAGG>hsa-mir-3176 MI0014210UGGCCUCUCCAGUCUGCAGCUCCCGGCAGCCUCGGGCCACACUCCCGGGAUCCCCAGGGACUGGCCUGGGACUACCGGGGGUGGCGGCCG>hsa-mir-3177 MI0014211CCACGUGCCAUGUGUACACACGUGCCAGGCGCUGUCUUGAGACAUUCGCGCAGUGCACGGCACUGGGGACACGUGGCACUGG>hsa-mir-3178 MI0014212GAGGCUGGGCGGGGCGCGGCCGGAUCGGUCGAGAGCGUCCUGGCUGAUGACGGUCUCCCGUGCCCACGCCCCAAACGCAGUCUC>hsa-mir-3179-1 MI0014213CAGGAUCACAGACGUUUAAAUUACACUCCUUCUGCUGUGCCUUACAGCAGUAGAAGGGGUGAAAUUUAAACGUCUGUGAUCCUG>hsa-mir-3179-2 MI0014216CAGGAUCACAGACGUUUAAAUUACACUCCUUCUGCUGUGCCUUACAGCAGUAGAAGGGGUGAAAUUUAAACGUCUGUGAUCCUG>hsa-mir-3179-3 MI0014221CAGGAUCACAGACGUUUAAAUUACACUCCUUCUGCUGUGCCUUACAGCAGUAGAAGGGGUGAAAUUUAAACGUCUGUGAUCCUG>hsa-mir-3180-1 MI0014214CAGUGCGACGGGCGGAGCUUCCAGACGCUCCGCCCCACGUCGCAUGCGCCCCGGGAAAGCGUGGGGCGGAGCUUCCGGAGGCCCCGCCCUGCUG>hsa-mir-3180-2 MI0014215GCGACGGGCGGAGCUUCCAGACGCUCCGCCCCACGUCGCAUGCGCCCCGGGAAAGCGUGGGGCGGAGCUUCCGGAGGCCCCGCCCUGC>hsa-mir-3180-3 MI0014217CAGUGCGACGGGCGGAGCUUCCAGACGCUCCGCCCCACGUCGCAUGCGCCCCGGGAAAGCGUGGGGCGGAGCUUCCGGAGGCCCCGCCCUGCUG>hsa-mir-3180-4 MI0016408GCUCCGCCCCACGUCGCAUGCGCCCCGGGAACGCGUGGGGCGGAGCUUCCGGAGGCCCCGCUCUGCUGCCGACCCUGUGGAGCGGAGGGUGAAGCCUCCGGAUGCCAGUCCCUCAUCGCUGGCCUGGUCGCGCUGUGGCGAAGGGGGCGGAGC>hsa-mir-3180-5 MI0016409GCUCCGCCCCACGUCGCAUGCGCCCCGGGAACGCGUGGGGCGGAGCUUCCGGAGGCCCCGCCCUGCUGCCGACCCUGUGGAGCGGAGGGUGAAGCCUCCGGAUGCCAGUCCCUCAUCGCUGGCCCGGUCGCGCUGUGGCGAAGGGGGCGGAGC>hsa-mir-3181 MI0014223CGGCGACCAUCGGGCCCUCGGCGCCGGCCCGUUAGUUGCCCGGGCCCGAGCCGGCCGGGCCCGCGGGUUGCCG>hsa-mir-3182 MI0014224GCUGCUUCUGUAGUGUAGUCCGUGCAUCCGCCCUUCGAUGCUUGGGUUGGAUCAUAGAGCAGU>hsa-mir-3183 MI0014225CUCUGCCCUGCCUCUCUCGGAGUCGCUCGGAGCAGUCACGUUGACGGAAUCCUCCGGCGCCUCCUCGAGGGAGGAGAGGCAGGG>hsa-mir-3184 MI0014226AAGCAAGACUGAGGGGCCUCAGACCGAGCUUUUGGAAAAUAGAAAAGUCUCGCUCUCUGCCCCUCAGCCUAACUU>hsa-mir-3185 MI0014227GAAUGGAAGAAGAAGGCGGUCGGUCUGCGGGAGCCAGGCCGCAGAGCCAUCCGCCUUCUGUCCAUGUC>hsa-mir-3186 MI0014229AGCCUGCGGUUCCAACAGGCGUCUGUCUACGUGGCUUCAACCAAGUUCAAAGUCACGCGGAGAGAUGGCUUUGGAACCAGGGGCU>hsa-mir-3187 MI0014231GCUGGCCCUGGGCAGCGUGUGGCUGAAGGUCACCAUGUUCUCCUUGGCCAUGGGGCUGCGCGGGGCCAGC>hsa-mir-3188 MI0014232GGCGCCUCCUGCUCUGCUGUGCCGCCAGGGCCUCCCCUAGCGCGCCUUCUGGAGAGGCUUUGUGCGGAUACGGGGCUGGAGGCCU>hsa-mir-3189 MI0014233GCCUCAGUUGCCCCAUCUGUGCCCUGGGUAGGAAUAUCCUGGAUCCCCUUGGGUCUGAUGGGGUAGCCGAUGC>hsa-mir-3190 MI0014235CUGGGGUCACCUGUCUGGCCAGCUACGUCCCCACGGCCCUUGUCAGUGUGGAAGGUAGACGGCCAGAGAGGUGACCCCGG>hsa-mir-3191 MI0014236GGGGUCACCUCUCUGGCCGUCUACCUUCCACACUGACAAGGGCCGUGGGGACGUAGCUGGCCAGACAGGUGACCCC>hsa-mir-3192 MI0014237GGAAGGGAUUCUGGGAGGUUGUAGCAGUGGAAAAAGUUCUUUUCUUCCUCUGAUCGCCCUCUCAGCUCUUUCCUUCU>hsa-mir-3193 MI0014238UCCUGCGUAGGAUCUGAGGAGUGGACGAGUCUCAUUACCCAGCUCCUGAGCAGGA>hsa-mir-3194 MI0014239AGGUGGCAGGGCCAGCCACCAGGAGGGCUGCGUGCCACCCGGGCAGCUCUGCUGCUCACUGGCAGUGUCACCU>hsa-mir-3195 MI0014240CCGCAGCCGCCGCGCCGGGCCCGGGUUGGCCGCUGACCCCCGCGGGGCCCCCGGCGGCCGGGGCGGGGGCGGGGGCUGCCCCGG>hsa-mir-3196 MI0014241GGGUGGGGGCGGGGCGGCAGGGGCCUCCCCCAGUGCCAGGCCCCAUUCUGCUUCUCUCCCAGCU>hsa-mir-3197 MI0014245GGCGAGGGGAGGCGCAGGCUCGGAAAGGCGCGCGAGGCUCCAGGCUCCUUCCCGAUCCACCGCUCUCCUCGCU>hsa-mir-3198-1 MI0014246GACUGUGCUCUCACUGUUCACCCAGCACUAGCAGUACCAGACGGUUCUGUGGAGUCCUGGGGAAUG
GAGAGAGCACAGUC>hsa-mir-3198-2 MI0017335GACUCUGCUCUCACUGUUCACCCAGCACUAGCAGUACCAGAUGGUUCUGUGGAGUCCUGGGGAAUGGAGAGAGCACAGUC>hsa-mir-3199-1 MI0014247GGUGACUCCAGGGACUGCCUUAGGAGAAAGUUUCUGGAAGUUCUGACAUUCCAGAAACUUUCUCCUAAGGCAGUCCCUGGGAGUCACU>hsa-mir-3199-2 MI0014248GUGACUCCCAGGGACUGCCUUAGGAGAAAGUUUCUGGAAUGUCAGAACUUCCAGAAACUUUCUCCUAAGGCAGUCCCUGGAGUCAC>hsa-mir-3200 MI0014249GGUGGUCGAGGGAAUCUGAGAAGGCGCACAAGGUUUGUGUCCAAUACAGUCCACACCUUGCGCUACUCAGGUCUGCUCGUGCCCU>hsa-mir-3201 MI0014250GGGAUAUGAAGAAAAAUAAGAGGCUAGGAUUGCCUCUUAUUUUUACAUGCCC>hsa-mir-3202-1 MI0014252UAUUAAUAUGGAAGGGAGAAGAGCUUUAAUGAUUGGAGUCAUUUUCAGAGCAUUAAAGCUCUUCUCCCUUCCAUAUUAAUG>hsa-mir-3202-2 MI0014253AUUAAUAUGGAAGGGAGAAGAGCUUUAAUGCUCUGAAAAUGACUCCAAUCAUUAAAGCUCUUCUCCCUUCCAUAUUAAU>hsa-mir-3529 MI0017351GGCACCAUUAGGUAGACUGGGAUUUGUUGUUGAGCGCAGUAAGACAACAACAAAAUCACUAGUCUUCCAGAUGGGGCC>hsa-mir-3545 MI0017343GCGCCCGCCGGGUCUAGUGGUCCUAAACAUUUCACAAUUGCGCUACAGAACUGUUGAACUGUUAAGAACCACUGGACCCAGCGCGC>hsa-mir-3591 MI0017383CAGUAGCUAUUUAGUGUGAUAAUGGCGUUUGAUAGUUUAGACACAAACACCAUUGUCACACUCCACAGCUCUG>hsa-mir-3605 MI0015995ACUUUAUACGUGUAAUUGUGAUGAGGAUGGAUAGCAAGGAAGCCGCUCCCACCUGACCCUCACGGCCUCCGUGUUACCUGUCCUCUAGGUGGGACGCUCG>hsa-mir-3606 MI0015996UUGUUGCUAUCUAGGUUAGUGAAGGCUAUUUUAAUUUUUUUAAAAUUUCUUUCACUACUUAGG>hsa-mir-3607 MI0015997AAGGUUGCGGUGCAUGUGAUGAAGCAAAUCAGUAUGAAUGAAUUCAUGAUACUGUAAACGCUUUCUGAUGUACUACUCA>hsa-mir-3609 MI0015999GUAACAGUAACUUUUAUUCUCAUUUUCCUUUUCUCUACCUUGUAGAGAAGCAAAGUGAUGAGUAAUACUGGCUGGAGCCC>hsa-mir-3610 MI0016000AAGAGCCGCGGCGUAACGGCAGCCAUCUUGUUUGUUUGAGUGAAUCGGAAAGGAGGCGCCGGCUGUGGCGGCG>hsa-mir-3611 MI0016001AGCAGGUCUAAUAAGAAUUUCUUUUUCUUCACAAUUAUGAAAGAAAAGAAAUUGUGAAGAAAGAAAUUCUUACUAGUUUUGCU>hsa-mir-3612 MI0016002GGGACUGGGGAUGAGGAGGCAUCUUGAGAAAUGGAAGGAAUGGGAUCUACUUCCAGUUCACUAGAGGCGUCCUGACACCCCUAGCUC>hsa-mir-3613 MI0016003UGGUUGGGUUUGGAUUGUUGUACUUUUUUUUUUGUUCGUUGCAUUUUUAGGAACAAAAAAAAAAGCCCAACCCUUCACACCACUUCA>hsa-mir-3614 MI0016004GGUUCUGUCUUGGGCCACUUGGAUCUGAAGGCUGCCCCUUUGCUCUCUGGGGUAGCCUUCAGAUCUUGGUGUUUUGAAUUCUUACU>hsa-mir-3615 MI0016005GACUCUGGGACGCUCAGACGCCGCGCGGGGCGGGGAUUGGUCUGUGGUCCUCUCUCGGCUCCUCGCGGCUCGCGGCGGCCGACGGUU>hsa-mir-3616 MI0016006UGUCACUCCGCCAGCAUCAUGAAGUGCACUCAUGAUAUGUUUGCCCCAUCAGCGUGUCACGAGGGCAUUUCAUGAUGCAGGCGGGGUUGGCA>hsa-mir-3617 MI0016007AGGUCAUAGAAAGACAUAGUUGCAAGAUGGGAUUAGAAACCAUAUGUCUCAUCAGCACCCUAUGUCCUUUCUCUGCCCU>hsa-mir-3618 MI0016008UAAGCUGAGUGCAUUGUGAUUUCCAAUAAUUGAGGCAGUGGUUCUAAAAGCUGUCUACAUUAAUGAAAAGAGCAAUGUGGCCAGCUUG>hsa-mir-3619 MI0016009ACGGCAUCUUUGCACUCAGCAGGCAGGCUGGUGCAGCCCGUGGUGGGGGACCAUCCUGCCUGCUGUGGGGUAAGGACGGCUGU>hsa-mir-3620 MI0016011GUGAGGUGGGGGCCAGCAGGGAGUGGGCUGGGCUGGGCUGGGCCAAGGUACAAGGCCUCACCCUGCAUCCCGCACCCAG>hsa-mir-3621 MI0016012GUGAGCUGCUGGGGACGCGGGUCGGGGUCUGCAGGGCGGUGCGGCAGCCGCCACCUGACGCCGCGCCUUUGUCUGUGUCCCACAG>hsa-mir-3622a MI0016013AAUAGAGGGUGCACAGGCACGGGAGCUCAGGUGAGGCAGGGAGCUGAGCUCACCUGACCUCCCAUGCCUGUGCACCCUCUAUU>hsa-mir-3622b MI0016014AGUGAUAUAAUAGAGGGUGCACAGGCAUGGGAGGUCAGGUGAGCUCAGCUCCCUGCCUCACCUGAGCUCCCGUGCCUGUGCACCCUCUAUUGGCU>hsa-mir-3646 MI0016046UUCAGUAGGUUGGGUUCAUUUCAUUUUCAUGACAACCCUAUAUGGGAAAAUGUUGUGAAAAUGAAAUGAGCCCAGCCCAUUGAA>hsa-mir-3648 MI0016048CGCGACUGCGGCGGCGGUGGUGGGGGGAGCCGCGGGGAUCGCCGAGGGCCGGUCGGCCGCCCCGGGUGCCGCGCGGUGCCGCCGGCGGCGGUGAGGCCCCGCGCGUGUGUCCCGGCUGCGGUCGGCCGCGCUCGAGGGGUCCCCGUGGCGUCCCCUUCCCCGCCGGCCGCCUUUCUCGCG>hsa-mir-3649 MI0016049GCUUGGAACAGGCACCUGUGUGUGCCCAAGUGUUUCUAGCAAACACAGGGACCUGAGUGUCUAAGC>hsa-mir-3650 MI0016050UCAAGGUGUGUCUGUAGAGUCCUGACUGCGUGCCAGGGGCUCUGUCUGGCACAUUUCUGA>hsa-mir-3651 MI0016051GAUUCGAUGGGCCAUAGCAAUCCUGUGAUUUAUGCAUGGAGGCUGCUUCUCCUCAGCAGCUGCCAUAGCCCGGUCGCUGGUACAUGAUUC>hsa-mir-3652 MI0016052CGGCUGGAGGUGUGAGGAUCCGAACCCAGGGGUGGGGGGUGGAGGCGGCUCCUGCGAUCGAAGGGGACUUGAGACUCACCGGCCGCACGCCAUGAGGGCCCUGUGGGUGCUGGGCCUCUGCUGCGUCCUGC>hsa-mir-3653 MI0016053UCCCUGGGGACCCCUGGCAGCCCCUCCUGAUGAUUCUUCUUCCUGAGCACGCUCAUGAUGAGCAAACUGAGCCUCUAAGAAGUUGACUGAAGGGGCUGCUUCCCCAAGGA>hsa-mir-3654 MI0016054UUCAUGAGCUGCAAUCUCAUCACUGGAAUGUUCCAGCGACUGGACAAGCUGAGGAA>hsa-mir-3655 MI0016055GCUUGUCGCUGCGGUGUUGCUGUUGGAGACUCGAUUGUUGGUGACAGCGAAAGAACGAUAACAAAAUGCCGGAGCGAGAUAGU>hsa-mir-3656 MI0016056CUUUCGGCCAGCGGGACGGCAUCCGAGGUGGGCUAGGCUCGGGCCCGUGGCGGGUGCGGGGGUGGGAGG>hsa-mir-3657 MI0016057UGUGUCCCAUAAUUAAAUAAUGAAAUCUGAAAUCACCAAUAAUGGGACACUAAUGUGAUUAAUGUUGUUGUGUCCCAUUAUUGGUGAUUUCAGAUUUCAUAUAUGAUUAAGGACAUA>hsa-mir-3658 MI0016058UAUUUAAGAAAACACCAUGGAGAUGAAAUGCCUUUGAUUUUUUUUUUCUUUUUGUA>hsa-mir-3659 MI0016060UCUACAAGCAGAUACAAGGAUGCCCUUGUACACAACACACGUGCUGCUUGUAUAGACAUGAGUGUUGUCUACGAGGGCAUCCUUGUGUCUGUGUGUGUG>hsa-mir-3660 MI0016061GAAAGAAGAACUGGACAAAAUUAAAAUGCUCUUCUGUCAUUGUAAUAGUUCAUAUGGGCACUGACAGGAGAGCAUUUUGACUUUGUCAAGUGUGUCUGCU>hsa-mir-3661 MI0016062CACCUUCUCGCAGAGGCUCUUGACCUGGGACUCGGACAGCUGCUUGCACUCGUUCAGCUGCUCGAUCCACUGGUCCAGCUCCUUGGUGAACACCUU>hsa-mir-3662 MI0016063UGUGUUUUCCUCAACGCUCACAGUUACACUUCUUACUCUCAAUCCAUUCAUAUUGAAAAUGAUGAGUAGUGACUGAUGAAGCACAAAUCAGCCAA>hsa-mir-3663 MI0016064CCCGGGACCUUGGUCCAGGCGCUGGUCUGCGUGGUGCUCGGGUGGAUAAGUCUGAUCUGAGCACCACACAGGCCGGGCGCCGGGACCAAGGGGGCUC>hsa-mir-3664 MI0016065CUGUAAACUUGAAGGUAGGGAACUCUGUCUUCACUCAUGAGUACCUUCCAACACGAGCUCUCAGGAGUAAAGACAGAGUUCCCUACCUUCAAUGUGGAU>hsa-mir-3665 MI0016066GCGGGCGGCGGCGGCGGCAGCAGCAGCAGGUGCGGGGCGGCGGCCGCGCUGGCCGCUCGACUCCGCAGCUGCUCGUUCUGCUUCUCCAGCUUGCGCACCAGCUCC>hsa-mir-3666 MI0016067AGUAAGGUCCGUCAGUUGUAAUGAGACCCAGUGCAAGUGUAGAUGCCGACUCCGUGGCAGAGUUCAGCGUUUCACACUGCCUGGUCUCUGUCACUCUAUUGAAUUAGAUUG>hsa-mir-3667 MI0016068UGAGGAUGAAAGACCCAUUGAGGAGAAGGUUCUGCUGGCUGAGAACCUUCCUCUCCAUGGGUCUUUCAUCCUCA>hsa-mir-3668 MI0016069AUAUAUGAAAUGUAGAGAUUGAUCAAAAUAGUUUCUAUCAAAAUAGUUUUGAUCAAUCUCUGCAAUUUUAUAUAU>hsa-mir-3669 MI0016070AUAUAUAUAUACGGAAUAUAUAUACGGAAUAUAUAUAUACGGAAUAUAUAUAUACGGAAUAUGUAUACGGAAUAUAUAUAU>hsa-mir-3670-1 MI0016071UCUAGACUGGUAUAGCUGCUUUUGGAGCCUCACCUGCUGAGAGCUCACAGCUGUCCUUCUCUAGA>hsa-mir-3670-2 MI0019112UCUAGACUGGUAUAGCUGCUUUUGGAGCCUCACCUGCUGAGAGCUCACAGCUGUCCUUCUCUAGA>hsa-mir-3671 MI0016072AUGUUAUUGCUGCUGCUGUCACAUUUACAUGAAAAUAAAAUGUAAAUUAUUUUAUUUCUAUCAAAUAAGGACUAGUCUGCAGUGAUAU>hsa-mir-3672 MI0016073UCUUUGUGAUUACCAUGAGACUCAUGUAAAACAUCUUAGACUAUUACAAGAUGUUUUAUGAGUCUCAUGAUAAUCACAAAGA>hsa-mir-3673 MI0016074AUAUAUAUAUAUGGAAUGUAUAUACGGAAUAUAUAUAUAUAUGGAAUGUAUAUACGGAAUAUAUAUAUAUAUGGAAUGUAUAUACGGAAUAUAUAUAUAUAU>hsa-mir-3674 MI0016075ACAUCACUAUUGUAGAACCUAAGAUUGGCCGUUUGAGAUGUCCUUUCAAGUUUUUGCAUUUCUGAUGU>hsa-mir-3675 MI0016076GGAUGAUAAGUUAUGGGGCUUCUGUAGAGAUUUCUAUGAGAACAUCUCUAAGGAACUCCCCCAAACUGAAUUC>hsa-mir-3676 MI0016077UUGGUUAAAGCGCCUGUCUAGUAAACAGGAGAUCCUGGGUUCGAAUCCCAGCGGUGCCUCCGUGUUUCCCCCACGCUUUUGCCAA>hsa-mir-3677 MI0016078GGCAGUGGCCAGAGCCCUGCAGUGCUGGGCAUGGGCUUCUCGUGGGCUCUGGCCACGGCC>hsa-mir-3678 MI0016079GAAUCCGGUCCGUACAAACUCUGCUGUGUUGAAUGAUUGGUGAGUUUGUUUGCUCAUUGAUUGAAUCACUGCAGAGUUUGUACGGACCGGAUUC>hsa-mir-3679 MI0016080CGUGGUGAGGAUAUGGCAGGGAAGGGGAGUUUCCCUCUAUUCCCUUCCCCCCAGUAAUCUUCAUCAUG>hsa-mir-3680-1 MI0016081AAAUUUAAGGAGGGACUCACUCACAGGAUUGUGCAAAUGCAAAGUUGGCUUUUGCAUGACCCUGGGAGUAGGUGCCUCCUUAAAUUU>hsa-mir-3680-2 MI0019113AAAUUUAAGGAGGGACUCACUCACAGGAUUGUGCAAAUGCAAAGUUGGCUUUUGCAUGACCCUGGGAGUAGGUGCCUCCUUAAAUUU>hsa-mir-3681 MI0016082ACUUCCAGUAGUGGAUGAUGCACUCUGUGCAGGGCCAACUGUGCACACAGUGCUUCAUCCACUACUGGAAGU>hsa-mir-3682 MI0016083UAAGUUAUAUAUGUCUACUUCUACCUGUGUUAUCAUAAUAAAGGUGUCAUGAUGAUACAGGUGGAGGUAGAAAUAUAUAACUUA>hsa-mir-3683 MI0016084GGGUGUACACCCCCUGCGACAUUGGAAGUAGUAUCAUCUCUCCCUUGGAUGCUACGAACAAUAUCACAGAAGGUGUACACCC>hsa-mir-3684 MI0016085AAUCUAAAGGACCUGUACUAGGUUUAACAUGUUGAGCAUUACUCAUGUUAGACCUAGUACACGUCCUUUAGAUU>hsa-mir-3685 MI0016086GUACAUUUCCUACCCUACCUGAAGACUUGAGAUUAUAGUCUUUGGGGGGAUGGGCAAAGUAC>hsa-mir-3686 MI0016087CUCACCUCAUUCAUUUACCUUCUCUUACAGAUCACUUUUCUGCACUGGACAGUGAUCUGUAAGAGAAAGUAAAUGAAAGAGGUGAG>hsa-mir-3687 MI0016088CGCGCGUGCGCCCGAGCGCGGCCCGGUGGUCCCUCCCGGACAGGCGUUCGUGCGACGUGUG>hsa-mir-3688-1 MI0016089UCUUCACUUUCAAGAGUGGCAAAGUCUUUCCAUAUGUAUGUAUGUAUGUCUGUUACACAUAUGGAAAGACUUUGCCACUCUUUAAAGUGAAGA>hsa-mir-3688-2 MI0017447UCACUUUAAAGAGUGGCAAAGUCUUUCCAUAUGUGUAACAGACAUACAUACAUACAUAUGGAAAGACUUUGCCACUCUUGAAAGUGA>hsa-mir-3689a MI0016090CCUGGGAGGUGUGAUAUCAUGGUUCCUGGGAGGUGUGAUCCUGUGCUUCCUGGGAGGUGUGAUAUCGUGGUUCCUGGG>hsa-mir-3689b MI0016411GAUCCUGUGCUCCCUGGGGGGUCUGAUCCUGUGCUUCCUGGGAGGUGUGAUAUCAUGGUUCCUGGGAGGUGUGAUCCCGUGCUUCCUGGGAGGUGUGAUAUUGUGGUUCCUGGGAGGUGUGAUCCCGUGCUCCCUGGGAGGUGUGAUC>hsa-mir-3689c MI0016832GGGAGGUGUGAUAUCGUGGUUCCUGGGAGGUGUGAUAUCGUGGUUCCUGGGAGGUGUGAUAUUGUGGUUCCU>hsa-mir-3689d-1 MI0016834UGGGAGGUGUGAUCUCACACUCGCUGGGAGGUGUGCUAUCGUCUUCCCCGGGAGGUGUGAUCCUGUUCUUCCUG>hsa-mir-3689d-2 MI0016835ACUGGGAGGUGUGAUCUCACACUCGCUGGGAGGUGUGCUAUCGUCUUCCCUGGGAGGUGUGAUCCUGUUCUUCCUGAGCG>hsa-mir-3689e MI0016836GGGAGGUGUGAUAUCAUGGUUCCUGGGAGGUAUGAUAUCGUGGUUCCUGGGAGGUGUGAUCCCGUGCUCCCU>hsa-mir-3689f MI0016837AGGUGUGAUAUCGUGCUUCCUGGGACGUGUGAUGCUGUGCUUCCUGGGAGGUGUGAUCCCACACUC>hsa-mir-3690 MI0016091CCCAUCUCCACCUGGACCCAGCGUAGACAAAGAGGUGUUUCUACUCCAUAUCUACCUGGACCCAGUGUAGAUGGG>hsa-mir-3691 MI0016092UUGAGGCACUGGGUAGUGGAUGAUGGAGACUCGGUACCCACUGCUGAGGGUGGGGACCAAGUCUGCGUCAUCCUCUCCUCAGUGCCUCAA>hsa-mir-3692 MI0016093CCAUUCCUGCUGGUCAGGAGUGGAUACUGGAGCAAUAGAUACAGUUCCACACUGACACUGCAGAAGUGG>hsa-mir-3713 MI0016134GGUAUCCGUUUGGGGAUGGUUUCACUAUCCCCAGAUGGAUACCAA>hsa-mir-3714 MI0016135GAAGGCAGCAGUGCUCCCCUGUGACGUGCUCCAUCACCGGGCAGGGAAGACACCGCUGCCACCUC>hsa-mir-3907 MI0016410GGGUUGGAAAGCUGUAGGUGUGGAGGGGCAUGGAUACGGGGGCCAUGAGGGUGGGGUCCAGGCUGGACCAGGCCUGCCCUGAGUCCCCCAGCAGGUGCUCCAGGCUGGCUCACACCCUCUGCCUCUCUCUCUUCCUUCCUGGCCCCAACCC>hsa-mir-3908 MI0016412GCCUGAGCAAUGUAGGUAGACUGUUUCUAAAAAAAUAAAAAGUUAAAAAAAUUUAUGUUAACGUGUAAUGUGUUUACUAAUUUUUUUUUUUUUUUUUGGAGACAGAGUCUCCCUCUGUCGCCAGGC>hsa-mir-3909 MI0016413GGUAUGCUGUUGCGCUGUCCUUCCUCUGGGGAGCAGGCUCCGGGGGACAGGGAAAAGCACACAAGGAACUUGUCCUCUAGGGCCUGCAGUCUCAUGGGAGAGUGACAUGCACCAGGACC>hsa-mir-3910-1 MI0016414CUUUUGCUGUCAGUUUUUCUGUUGCUUGUCUUGGUUUUAUGCCUUUUAUAUCAAGGCACAUAAAAGGCAUAAAACCAAGACAAGCAACAAAAAAAGGAUUGAUCACAGAAG>hsa-mir-3910-2 MI0016431UUUUUUUGUUGCUUGUCUUGGUUUUAUGCCUUUUAUGUGCCUUGAUAUAAAAGGCAUAAAACCAAGACAAGCAACAGAAAAA>hsa-mir-3911 MI0016415GGGUGAGGAUGUGUGUGGAUCCUGGAGGAGGCAGAGAAGACAGUGAGCUUGCCAGUUCUGGUUUCCAACACUUCCUUUCCUGCGCUUCUCGAUUCCCAGAUCUGCACCC>hsa-mir-3912 MI0016416AGAGAGGA
AUGAACAGUUAAAUUAUAACAUGUCCAUAUUAUGGGUUAGUUGUGGACACAUACUAACGCAUAAUAUGGACAUGUUAUAAUUUAACUGUUCCUUUCU>hsa-mir-3913-1 MI0016417UUGUUUAUAAUAAACUGAAAUAUUUGGGACUGAUCUUGAUGUCUGCCAAAACCUUGGCAGACAUCAAGAUCAGUCCCAAAUAUUUCAGUUUAUUAUAGACAG>hsa-mir-3913-2 MI0016418UGUCUAUAAUAAACUGAAAUAUUUGGGACUGAUCUUGAUGUCUGCCAAGGUUUUGGCAGACAUCAAGAUCAGUCCCAAAUAUUUCAGUUUAUUAUAAACA>hsa-mir-3914-1 MI0016419UGGACUUCAGAUUUAACUUCUCAUUUUCUGGUUCCUUCUAAUGAGUAUGCUUAACUUGGUAGAAGGAACCAGAAAAUGAGAAGUUGAGUAGGAACUCUA>hsa-mir-3914-2 MI0016421GAGUUCCUACUCAACUUCUCAUUUUCUGGUUCCUUCUACCAAGUUAAGCAUACUCAUUAGAAGGAACCAGAAAAUGAGAAGUUAAAUCUGAAGUC>hsa-mir-3915 MI0016420CAAGUUGGCACUGUAGAAUAUUGAGGAAAAGAUGGUCUUAUUGCAAAGAUUUUCAAUAAGACCAUCCUUUCCUCAAUAUUCUGUGGUGUCAUCUUUG>hsa-mir-3916 MI0016422AUCCCAGAGAAGAAGGAAGAAGAGGAAGAAAUGGCUGGUUCUCAGGUGAAUGUGUCUGGGUUCAGGGGAUGUGUCUCCUCUUUUCUUCUGGGAU>hsa-mir-3917 MI0016423GGCGCUUUUGUGCGCGCCCGGGUCUGUUGGUGCUCAGAGUGUGGUCAGGCGGCUCGGACUGAGCAGGUGGGUGCGGGGCUCGGAGGAGGCGGC>hsa-mir-3918 MI0016424AGGCGGUUAAGCCAUGGGACAGGGCCGCAGAUGGAGACUGCUCAAGGUCAAAGGGGUCUCCAGCUGGGACCCUGCACCUGGUUCGUAGCCCCU>hsa-mir-3919 MI0016425CCUGAGCACCAUUUACUGAGUCCUUUGUUCUCUACUAGUUUGUAGUAGUUCGUAGCAGAGAACAAAGGACUCAGUAAAUGGUGCUCAGG>hsa-mir-3920 MI0016427ACUGAGUGAGGGAGUCAGAGAGUUAAGAGAAUUAGUACAGGUGAGAUUGUACUGAUUAUCUUAACUCUCUGACCCCCUCACUCAGU>hsa-mir-3921 MI0016428CCUAGCCCAGUACAAGGCAUAUGGUACUCAAGAGACUUAGAAAUCCCUAAGUCUCUGAGUACCAUAUGCCUUGUACUGGGCUAGG>hsa-mir-3922 MI0016429GGAAGAGUCAAGUCAAGGCCAGAGGUCCCACAGCAGGGCUGGAAAGCACACCUGUGGGACUUCUGGCCUUGACUUGACUCUUUC>hsa-mir-3923 MI0016430GGUAGAGUGAGCUCUAAUCCAAUAUUACUAGCUUCUUUAUAAGAAGAGGAAACUAGUAAUGUUGGAUUAGGGCUCACUCUACU>hsa-mir-3924 MI0016432UAAAUGAAAAAGUAGUAGUCAAAUAUGCAGAUCUAUGUCAUAUAUACAGAUAUGUAUAUGUGACUGCUACUUUUUUGUUUA>hsa-mir-3925 MI0016433GUGGGAAUAGCAAGAGAACUGAAAGUGGAGCCUGUCACAUCUCCAGACUCCAGUUUUAGUUCUCUUGCUAUUUCCAC>hsa-mir-3926-1 MI0016434AAAAUGGAGCUGGCCAAAAAGCAGGCAGAGACUUUAAAAGCGUCUCUGCCUGCUUUUUGGCCAGCUCCGUUUU>hsa-mir-3926-2 MI0016437GGAGCUGGCCAAAAAGCAGGCAGAGACGCUUUUAAAGUCUCUGCCUGCUUUUUGGCCAGCUCC>hsa-mir-3927 MI0016435UGCCAAUGCCUAUCACAUAUCUGCCUGUCCUAUGACAAACAUGGCAGGUAGAUAUUUGAUAGGCAUUGGCA>hsa-mir-3928 MI0016438GCUGAAGCUCUAAGGUUCCGCCUGCGGGCAGGAAGCGGAGGAACCUUGGAGCUUCGGC>hsa-mir-3929 MI0016439AGUGGCUCACACCAGUAAUCCCAGCACUUUGGGAGGCUGAUGUGAGUAGACCACU>hsa-mir-3934 MI0016590CACAGCCCUUCCUGUCCCCAGUUUUCAGGUGUGGAAACUGAGGCAGGAGGCAGUGAAGUAACUUGCUCAGGUUGCACAGCUGGGAAGUGGAGCAGGGAUUUGAAUCC>hsa-mir-3935 MI0016591GGAUGUGUUCCUGUCCCAGAAGGAGCUGAUGGUUGUAUCUAUGAAGGUAAGCAUUUUUGUAGAUACGAGCACCAGCCACCCUAAGCAAAGGCAGAGAAUGCUUA>hsa-mir-3936 MI0016592AUGAUUCAGAGCAUCUGUCCAGUGUCUGCUGUAGAUCCCUCAAAUCCGUGUUUGGACGCUUCUGGUAAGGGGUGUAUGGCAGAUGCACCCGACAGAUGCACUUGGCAGCA>hsa-mir-3937 MI0016593AGAAGAAUGCCCAACCAGCCCUCAGUUGCUACAGUUCCCUGUUGUUUCAGCUCGACAACAACAGGCGGCUGUAGCAAUGGGGGGCUGGAUGGGCAUCUCAAUGUGC>hsa-mir-3938 MI0016594AGGAAUUUUUAACCCGAUCACUAGAUUAUCUACAAGGGAAUUUUUUUUUAAUUUAAAAAAUUCCCUUGUAGAUAACCCGGUGGUCAGGUUGGAUGGCUCCAUG>hsa-mir-3939 MI0016596CUGGCUUCCAAAGGCCUCUGUGUGUUCCUGUAUGUGGGCGUGCACGUACCUGUCACAUGUGUACGCGCAGACCACAGGAUGUCCACACUGGCUUCCAAACACAUCU>hsa-mir-3940 MI0016597GCUUAUCGAGGAAAAGAUCGAGGUGGGUUGGGGCGGGCUCUGGGGAUUUGGUCUCACAGCCCGGAUCCCAGCCCACUUACCUUGGUUACUCUCCUUCCUUCU>hsa-mir-3941 MI0016598GAGUCAGAAUUCUCAUCAGGCUGUGAUGCUCAGUUGUGUGUAGAUUGAAAGCCCUAAUUUUACACACAACUGAGGAUCAUAGCCUGAUGGUUCCUUUUUGUUU>hsa-mir-3942 MI0016599UCUUCAGUAUGACACCUCAAAGAAGCAAUACUGUUACCUGAAAUAGGCUGCGAAGAUAACAGUAUUUCAGAUAACAGUAUUACAUCUUUGAAGUGUCAUAUUCACUGAC>hsa-mir-3943 MI0016600CACACAGACGGCAGCUGCGGCCUAGCCCCCAGGCUUCACUUGGCGUGGACAACUUGCUAAGUAAAGUGGGGGGUGGGCCACGGCUGGCUCCUACCUGGAC>hsa-mir-3944 MI0016601UCCACCCAGCAGGCGCAGGUCCUGUGCAGCAGGCCAACCGAGAAGCGCCUGCGUCUCCCAUUUUCGGGCUGGCCUGCUGCUCCGGACCUGUGCCUGAUCUUAAUGCUG>hsa-mir-3945 MI0016602GAUGUUGAUGCACGUGACGGGGAGGGCAUAGGAGAGGGUUGAUAUAAAAUGCAAUUACAGCCUCUUAUGCUUUCCAAAGUGGGGGAUGAUUCAAUGAU>hsa-mir-3960 MI0016964GGCGCCCCGGCUCCCCGCGCCCCCGAUCGGGGCCGCCGCUAGUAGUGGCGGCGGCGGAGGCGGGGGCAGCGGCGGCGGCGGCGGAGGCGCC>hsa-mir-3972 MI0016990GCCCAUUUGCCUUGGCUUGGGGUGGCAGUCCUGUGGGAAUGAGAGAUGCCAAACUGGACCUGCCAGCCCCGUUCCAGGGCACAGCAU>hsa-mir-3973 MI0016991GCCCAGGGUAGCUCUCUGUAUUGCUUGUUACAUUUUAGGAUUGCUUGCCCCUUGCUCCAAUGGUGCAGGCAGAAGAAAUGCAAACAAAGUACAGCAUUAGCCUUAGC>hsa-mir-3974 MI0016992GUUCAGGGAAAAGGUCAUUGUAAGGUUAAUGCACCCAUAUUUUAAUAUCAAACUAUGACAAAUUUGACUACAGCCUUUCCGUACCCCUGCCAAAAC>hsa-mir-3975 MI0016993CAUGUAGGUGAGUGAUUGCUAUUUCAAAAGACUAGAGGUAGGAAAUGAGGCUAAUGCACUACUUCACAUG>hsa-mir-3976 MI0016994UGAGGGAUAUAGAGAGCAGGAAGAUUAAUGUCAUAUUGGAGUUGGACUGCAGGGCUUCCUUUACACAAUAAAUAUUGUAUGAAGUGCUGAUGUAACCUUUACUGCAGCAUGACAUGGGAUUUGGCUGUUUUUAUGGCUC>hsa-mir-3977 MI0016995UUGUGCUUCAUCGUAAUUAACCUUAAGUGGUUCGGGUAAAUCACUUUAAUUUGUUAUGUGUUGGCAGAAU>hsa-mir-3978 MI0016996UCAGUGGAAAGCAUGCAUCCAGGGUGUGGAGCCAAAAUUAGAAGGGCCAAAAUUCUACCUGGCCCACUACCACAGCAACCUUGGGCAUCGUUUUCUUUUGA>hsa-mir-4251 MI0015861CACGUCCUCCAGCUUUUUUCCUUAGUGGCCAAUUCCUGAGAAAAGGGCCAACGUGCUUCCA>hsa-mir-4252 MI0015864UGGGGGGCUGGCAGCUCAUCAGUCCAGGCCAUCUGGCCACUGAGUCAGCACCAGCGCCCAAUC>hsa-mir-4253 MI0015860CCAGCCAUCGCCCUUGAGGGGCCCUAGGACUUACUUGUGCAGGGCAUGUCCAGGGGGUCCAGGUCUGC>hsa-mir-4254 MI0015862CUUGGGAGGAGGGUGGGGUGGCUCCUCUGCAGUGAGUAGGUCUGCCUGGAGCUACUCCACCAUCUCCCCCAGCCCC>hsa-mir-4255 MI0015863GAGCAUCCUUCAGUGUUCAGAGAUGGAGUCAGUAUUGGUCUGGCCAUUUUUAGGGCAAAGAGGCAGCAUCAU>hsa-mir-4256 MI0015855UGUUCCAUUUAUCUGACCUGAUGAAGGUCUCCUGGCAUUGAUUAGGUCUGAUGAUCCAUUUCUG>hsa-mir-4257 MI0015856GGCUUAGAAACAGUCCCUAGGUAGGAUUUGGGGAGGAGCUAAGAAGCCCCUACAGGGCCCAGAGGUGGGGACUGAGCCUUAGUUGG>hsa-mir-4258 MI0015857ACGCCCCCCGCCCCGCCACCGCCUUGGAGGCUGACCUCUUACUUUCGGUCGGUCUUCUUCCCUGGGCUUGGUUUGGGGGCGGGGGAGUGUC>hsa-mir-4259 MI0015858GAUGGGCCCCUUGUGUCCUGAAUUGGGUGGGGGCUCUGAGUGGGGAAAGUGGGGGCCUAGGGGAGGUCACAGUUGGGUCUAGGGGUCAGGAGGGCCCAGGA>hsa-mir-4260 MI0015859AACAAGGUGACUUGGGGCAUGGAGUCCCACUUCCUGGAGCCCACACCCCAGCUUGUCACACACCAAC>hsa-mir-4261 MI0015868GGUGGAAGUGGGUUCCUCCCAGUUCCUGAGACAGGAAACAGGGACCCAGGAGACCAGC>hsa-mir-4262 MI0015872GAAAGCUGCAGGUGCUGAUGUUGGGGGGACAUUCAGACUACCUGCAGCAGAGCC>hsa-mir-4263 MI0015876AUAGUGCUCUUCAGGGUUUUACUUGGGAGAUUGGAGUGGCCAGUGUUCCUAAACAAUUCUAAGUGCCUUGGCCCACAACAUAC>hsa-mir-4264 MI0015877AAAGCUGGAUACUCAGUCAUGGUCAUUGUAACAUGAUAGUGACAGGUACUGGGUAAGACUGCAUAG>hsa-mir-4265 MI0015869UGCAGUGGGUUGGAGCUUCAGCCUACACCUGUAAAGAAUUGGUCAGCCUGGGGACUGGUGAUCUCUGCAGCUGUGGGCUCAGCUCUGGGCUGGGCCUGG>hsa-mir-4266 MI0015870CCACUGCUGGCCGGGGCCCCUACUCAAGGCUAGGAGGCCUUGGCCAAGGACAGUC>hsa-mir-4267 MI0015871CUCAGCAGGCUCCAGCUCGGUGGCACUGGGGGAAGGCUCCAGACCCCAGCCUCUGUCAUCCCUGCAUGGAGCCCACAUCUCC>hsa-mir-4268 MI0015874AUGCACAUCAGGUUCUAGAGGUUUUGCCCUAGCGGCUCCUCCUCUCAGGAUGUGAUGUCACCUG>hsa-mir-4269 MI0015875ACAGCGCCCUGCAGGCACAGACAGCCCUGGCUUCUGCCUCUUUCUUUGUGGAAGCCACUCUGUCAGGCCUGGGAUGGAGGGGCA>hsa-mir-4270 MI0015878ACAAAUAGCUUCAGGGAGUCAGGGGAGGGCAGAAAUAGAUGGCCUUCCCCUGCUGGGAAGAAAGUGGGUC>hsa-mir-4271 MI0015879AAAUCUCUCUCCAUAUCUUUCCUGCAGCCCCCAGGUGGGGGGGAAGAAAAGGUGGGGAAUUAGAUUC>hsa-mir-4272 MI0015880UUUUCUGCACAAAUUAAUCAGUUAAUGCAUAGAAAGCAUUCAACUAGUGAUUGUGUUAUAAGAG>hsa-mir-4273 MI0015881UCCCCUGUGUGUGUUCUCUGAUGGACAGUAAGCCUUGACUUAUGGCUAAAUGCUUCUUCACAAUGGUCACAUGCAUAGGGCUUU>hsa-mir-4274 MI0015884GGGGCAUUUAGGGUAACUGAGCUGCUGCCGGGGCCUGGCGCUCCUCUACCUUGUCAGGUGACCCAGCAGUCCCUCCCCCUGCAUGGUGCCC>hsa-mir-4275 MI0015883ACAUUUUUGUCCAAUUACCACUUCUUUUUGCCACCUGAGCACAGUCAGCAGUCAGCAUAAAAAAGUGAUAAUGGGAAGUUAAUGUCU>hsa-mir-4276 MI0015882CACAGUCUGACUCAGUGACUCAUGUGCUGGCAGUGGCCACGUAAAUAGAGCUACUGUGUCUGAAAGCAAU>hsa-mir-4277 MI0015886CUGGGUCGAGGCAGUUCUGAGCACAGUACACUGGGCUGCCCCCACUGCCCAGUGCCCUGCUCAGCUCAAGUCCUUGUGCCCCUC>hsa-mir-4278 MI0015888AUCUAACACCAGGAGAAUCCCAUAGAACAUUGACAUCAACACUAGGGGGUUUGCCCUUGUGGGGAAGAA>hsa-mir-4279 MI0015887UGCUCUGUGGAGCUGAGGAGCAGAUUCUCUCUCUCUCCUCCCGGCUUCACCUCCUGAG>hsa-mir-4280 MI0015889AAUCAGGGUGGAGUGUAGUUCUGAGCAGAGCCUUAAAGGAUGAGGUAUGUUCAAGACUGAAUGACACCUUUGUGAU>hsa-mir-4281 MI0015885GCUGGGGGUCCCCCGACAGUGUGGAGCUGGGGCCGGGUCCCGGGGAGGGGGGUUCUGGGCAG>hsa-mir-4282 MI0015890GGUGAAGUUCCAGGGGAAGAUUUUAGUAUGCCACAUUUCUAAAAUUUGCAUCCAGGAACAUCAUCCU>hsa-mir-4283-1 MI0015892ACUCUGAUCCUGGGGCUCAGCGAGUUUGCAAGGGGUGUUUCUGUCCAUGGUCAGGCUUGCCAGCCUUGGUCCUUGGGCCC>hsa-mir-4283-2 MI0015982ACUCUGAUCCUGGGGCUCAGCGAGUUUGCAAGGGGUGUUUCUGUCCAUGGUCAGGCUUGCCAGCCUUGGUCCUUGGGCCC>hsa-mir-4284 MI0015893GUUCUGUGAGGGGCUCACAUCACCCCAUCAAAGUGGGGACUCAUGGGGAGAGGGGGUAGUUAGGAGCUUUGAUAGAGGCGG>hsa-mir-4285 MI0015891AUUAGCUGGGGCGGCGAGUCCGACUCAUCAAUAUUUUAAGGAAUGACCCGGCCUUGGGGUGCGGAAUUGCUGCGCGGGCGGGGGC>hsa-mir-4286 MI0015894UACUUAUGGCACCCCACUCCUGGUACCAUAGUCAUAAGUUAGGAGAUGUUAGAGCUGUGAGUACCAUGACUUAAGUGUGGUGGCUUAAACAUG>hsa-mir-4287 MI0015895UAGUUCUUUUUCUCCCUUGAGGGCACUUUUCAGUUCCUGAGAUCAAUGUGGUCCCUACUGGGGAGACCAUAGGAGCCC>hsa-mir-4288 MI0015896AUGGAGGUGGAGAGUCAUCAGCAGCACUGAGCAGGCAGUGUUGUCUGCUGAGUUUCCACGUCAUUUG>hsa-mir-4289 MI0015898CCUUGGGAGGGCAUUGUGCAGGGCUAUCAGGCAGUUUCCUGGGCCCUGUCUGCAGAGCCUAAACAGAUCA>hsa-mir-4290 MI0015899GCCACCAAGAAGGUGAAGGGAGGGUCAGUCCCAAUCUGAAUCCCACCAAAAUAGGUGGUAGAGGGUUGCCCUCCUUUCUUCCCUCACCUCUGACC>hsa-mir-4291 MI0015900CGCCGGGGGCUUCAGCAGGAACAGCUGGGUGGAGGCAGAGCUGUUCUGCUGUGGCUGCAGCCCUG>hsa-mir-4292 MI0015897GAGACACCAGAAGGCCACCUGCUUAGGAGGCCAGAGGUGCCCCUGGGCCGGCCUUGGUGAGGGGCCC>hsa-mir-4293 MI0015826AGAGACACCUGUUCCUUGGGAAGCUGGUGACAUUGCUAAUUCAUUUCACACCAGCCUGACAGGAACAGCCUGACUGAA>hsa-mir-4294 MI0015827CCGAUGCCUCGGGAGUCUACAGCAGGGCCAUGUCUGUGAGGGCCCAAGGGUGCAUGUGUCUCCCAGGUUUCGGUGC>hsa-mir-4295 MI0015822CUUUGUGGAACAGUGCAAUGUUUUCCUUGCCUGUGGCAAGACCACUUCGGUUCAAGGCUAAGAAACUAGACUGUUCCUACAGAGA>hsa-mir-4296 MI0015823UUGGGCUUUGAUGUGGGCUCAGGCUCAGAGGGCUGAAGUGGUUGUGGGGAGGGGCUUCUGGGGACUGUGUCCAUGUCUCUGUCGUUUU>hsa-mir-4297 MI0015824AGCACGCACGUGCCUUCCUGUCUGUGCCUGCCUUCGAAGUGCACGGCAGGGCCAGGACGGGUCGCUGUGGGUGGGG>hsa-mir-4298 MI0015830GGGGAGGUACCUGGGACAGGAGGAGGAGGCAGCCUUGCCUCAGAAACCAAACUGUCAAAAGUGUAGGUUCCAC>hsa-mir-4299 MI0015829GGGUUCUGACCAAUCAUGUUACAGUGUUUUCUCCUUUAGAGAGAGCUGGUGACAUGAGAGGCAGAAAAAGGA>hsa-mir-4300 MI0015831UGAGUUUAGAAGAGGGCCAGCUAAAUCAGCAGAGACAUGAGGUGAUCAAAAACCUUUUUUCAAAGCAGUGGGAGCUGGACUACUUCUGAACCAAUA>hsa-mir-4301 MI0015828ACCAGCCACCUCCCACUACUUCACUUGUGAACAUUGCAUUCGUGGAGGGUGGCAGGUGCAGCUCUG>hsa-mir-4302 MI0015833UCAGGAGGGACCAGUGUGGCUCAGCGAGGUGGCUGAGUUUACUUAAGGUAUUGGAAUGAG>hsa-mir-4303 MI0015834AGAAAAUAGCUUCUG
AGCUGAGGACAGCUUGCUCUGCUUUUCUUUAGCUUAGGAGCUAACCAUGGU>hsa-mir-4304 MI0015832AGAGAAGUGGCCGGCAUGUCCAGGGCAUCCCCAUUGCUCUGUGACUGCUGCCAUCCUUCUCC>hsa-mir-4305 MI0015835CUGCCUUAGACCUAGACACCUCCAGUUCUGGGUUCUUAGAGGCCUAAUCCUCUACAAACUCAGUUUUCAGACUGUGAGGGAAAUUCUCUGUCUUAUUGCUUU>hsa-mir-4306 MI0015836AAGCUGCUUAGUGUCCUUAGAGUCUCCAGAGGCAUCCCUAACCCAGAAUCUUUUGACUGUCCUCUGGAGAGAAAGGCAGUAGGUCUGUACC>hsa-mir-4307 MI0015838UCAGAAGAAAAAACAGGAGAUAAAGUUUGUGAUAAUGUUUGUCUAUAUAGUUAUGAAUGUUUUUUCCUGUUUCCUUCAGGGCCA>hsa-mir-4308 MI0015839UAUGGGUUCAGAGGGAACUCCAUUGGACAGAAAUUUCCUUUUGAGGAAAUCUUUCCCUGGAGUUUCUUCUUACCUUUUUCC>hsa-mir-4309 MI0015837UCUGGGGGUUCUGGAGUCUAGGAUUCCAGGAUCUGGGUUUUGAGGUCUUGGGUUGUAGGGUCUGCGGUUUGAAGCCCCUCUUG>hsa-mir-4310 MI0015840UGGCGUCUGGGGCCUGAGGCUGCAGAACAUUGCAGCAUUCAUGUCCCACCCCCACCA>hsa-mir-4311 MI0015841UCAGAGAGGGGAAAGAGAGCUGAGUGUGACCUGGAGCAGCUCAGGAGGGCUUCCUGGGUGAGGUGGCAGGUUACAGGUUCGAUCUUUGGCCCUCAGAUUC>hsa-mir-4312 MI0015842GAAAGGUUGGGGGCACAGAGAGCAAGGAGCCUUCCCCAGAGGAGUCAGGCCUUGUUCCUGUCCCCAUUCCUCAGAG>hsa-mir-4313 MI0015843GAUCAGGCCCAGCCCCCUGGCCCCAAACCCUGCAGCCCCAGCUGGAGGAUGAGGAGAUGCUGGGCUUGGGUGGGGGAAUCAGGGGUGUAAAGGGGCCUGCU>hsa-mir-4314 MI0015846GGCCAUUCCUCUCUGGGAAAUGGGACAGGUAGUGGCCACAGUGAGAAAGCUGGCCUGUCCUUCUGCCCCAGGGCCCAGAGUCUGUGACUGGA>hsa-mir-4315-1 MI0015844UGGGCUUUGCCCGCUUUCUGAGCUGGACCCUCUCUCUACCUCUGGUGCAGAACUACAGCGGAAGGAAUCUCUG>hsa-mir-4315-2 MI0015983UGGGCUUUGCCCGCUUUCUGAGCUGGACCCUCUCUCUACCUCUGGUGCAGAACUACAGCGGAAGGAAUCUCUG>hsa-mir-4316 MI0015845AGUGGCCCAGGGUGAGGCUAGCUGGUGUGGUCACCCACUCUCCAGCCCAGCCCCAAUCCCACCACAACCAC>hsa-mir-4317 MI0015850AAAAGGCGAGACAUUGCCAGGGAGUUUAUUUUGUAGCUCUCUUGAUAAAAUGUUUUAGCAAACAC>hsa-mir-4318 MI0015847GCUUCUUAAUUAUGUCAUAAACCCACUGUGGACAAGGGCCUUGUCUUAGACAGUCACUGUGGGUACAUGCUAGGUGCUCAA>hsa-mir-4319 MI0015848UUGGCUUGAGUCCCUGAGCAAAGCCACUGGGAAUGCUCCCUGAGGACGUUAUAUGAGUGCUCAGCUCAUGGGGCUAUGAUGGUCA>hsa-mir-4320 MI0015849GACAUGUGGGGUUUGCUGUAGACAUUUCAGAUAACUCGGGAUUCUGUAGCUUCCUGGCAACUUUG>hsa-mir-4321 MI0015852CUGGUCUCCGCAGAGCCUCUGCCCCUCCCGAGACACCCGCUACCUGGUGUUAGCGGUGGACCGCCCUGCGGGGGCCUGGC>hsa-mir-4322 MI0015851ACCGCGAGUUCCGCGCCUGGCCGUGUCGCCCCACGAGGGGGACUGUGGGCUCAGCGCGUGGGGCCCGGAGCAU>hsa-mir-4323 MI0015853CGGGGCCCAGGCGGGCAUGUGGGGUGUCUGGAGACGCCAGGCAGCCCCACAGCCUCAGACCUCGGGCAC>hsa-mir-4324 MI0015854CGGCCCCUUUGUUAAGGGUCUCAGCUCCAGGGAACUUUAAAACCCUGAGACCCUAACCUUAAAGGUGCUGCA>hsa-mir-4325 MI0015865GGGGAAGAUGUUGCACUUGUCUCAGUGAGAGAUGCUUCUAGAUCCAGGAGGCAGACCUCAAGGAUGGAGAGAAGGCAGAUCCUUUGAGAU>hsa-mir-4326 MI0015866GCUGCUCUGCUGUUCCUCUGUCUCCCAGACUCUGGGUGGAUGGAGCAGGUCGGGGGCCA>hsa-mir-4327 MI0015867GGCCUGGGUAGGCUUGCAUGGGGGACUGGGAAGAGACCAUGAACAGGUUAGUCCAGGGAGUUCUCAUCAAGCCUUUACUCAGUAG>hsa-mir-4328 MI0015904AACAGUUGAGUCCUGAGAACCAUUGAGAACCAGUUUUCCCAGGAUUAACUGUUCCG>hsa-mir-4329 MI0015901UAGAGAGGAAGGUGUACCAGGGUUUUGGAGUUUUUUUUUCCUCCUGAGACCCUAGUUCCACAUUCUGGAGC>hsa-mir-4330 MI0015902AAUUGUCAGCAGGCAAUUAUCUGAGGAUGCAGGAGAGGAAGGGGGCUUCUUUUUGACGCCUACUUCAUCAGCUGCUCCUCAGAUCAGAGCCUUGCAGGUCAGGCC>hsa-mir-4417 MI0016753GAAAACAACCAGGUGGGCUUCCCGGAGGGCGGAACACCCAGCCCCAGCAUCCAGGGCUCACCUACCACGUUUG>hsa-mir-4418 MI0016754UGGUUUUUGCUCUGAGUGACCGUGGUGGUUGUGGGAGUCACUGCAGGACUCAGCAGGAAUUC>hsa-mir-4419a MI0016755UGGUGGUGUGUGCCUGUAGUCUUAGCUACUCGGGAGGCUGAGGGAGGAGACUGCAGUGAGUGGAGGUCACGCCACUG>hsa-mir-4419b MI0016861CUCAGGCUCAGUGGUGCAUGCUUAUAGUCCCAGCCACUCUGGAGGCUGAAGGAAGAUGGCUUGAGCCU>hsa-mir-4420 MI0016757CUCUUGGUAUGAACAUCUGUGUGUUCAUGUCUCUCUGUGCACAGGGGACGAGAGUCACUGAUGUCUGUAGCUGAGAC>hsa-mir-4421 MI0016758CUGGGUCUCCUUUCUGCUGAGAGUUGAACACUUGUUGGGACAACCUGUCUGUGGAAAGGAGCUACCUAC>hsa-mir-4422 MI0016759AGUUCUUCUGCAGACAAAAGCAUCAGGAAGUACCCACCAUGUACCAGUGGGCCCUUCUUGAUGCUCUUGAUUGCAGAGGAGCC>hsa-mir-4423 MI0016760AUCAUGUACUGCAGUUGCCUUUUUGUUCCCAUGCUGUUUAAGCCUAGCAUAGGCACCAAAAAGCAACAACAGUAUGUGAA>hsa-mir-4424 MI0016763CUUACAUCACACACAGAGUUAACUCAAAAUGGACUAAUUUUUCCACUAGUUAGUCCAUUUCAAGUUAACUCUGUGUGUGAUGUAGU>hsa-mir-4425 MI0016764GUGCUUUACAUGAAUGGUCCCAUUGAAUCCCAACAGCUUUGCGAAGUGUUGUUGGGAUUCAGCAGGACCAUUCGUGUAAAGUAA>hsa-mir-4426 MI0016765AGUUGGAAGAUGGACGUACUUUGUCUGACUACAAUAUUCAAAAGGAGUCUACUCUUCAUCUUG>hsa-mir-4427 MI0016766GAAGCCUCUUGGGGCUUAUUUAGACAAUGGUUUCAUCAUUUCGUCUGAAUAGAGUCUGAAGAGUCUUU>hsa-mir-4428 MI0016767UUGGCAGGUGCCAUGUUGCCUGCUCCUUACUGUACACGUGGCUGGCAAGGAGACGGGAACAUGGAGCCGCCAU>hsa-mir-4429 MI0016768AGGGAGAAAAGCUGGGCUGAGAGGCGACUGGUGUCUAAUUUGUUUGUCUCUCCAACUCAGACUGCCUGGCCCA>hsa-mir-4430 MI0016769GUGAGGCUGGAGUGAGCGGAGAUCGUACCACUGCACUCCAACCUGGUGA>hsa-mir-4431 MI0016771UGGUUUGCGACUCUGAAAACUAGAAGGUUUAUGACUGGGCAUUUCUCACCCAAUGCCCAAUAUUGAACUUUCUAGUUGUCAGAGUCAUUAACCC>hsa-mir-4432 MI0016772GCAUCUUGCAGAGCCGUUCCAAUGCGACACCUCUAGAGUGUCAUCCCCUAGAAUGUCACCUUGGAAAGACUCUGCAAGAUGCCU>hsa-mir-4433 MI0016773CAUCCUCCUUACGUCCCACCCCCCACUCCUGUUUCUGGUGAAAUAUUCAAACAGGAGUGGGGGUGGGACAUAAGGAGGAUA>hsa-mir-4434 MI0016774UCACUUUAGGAGAAGUAAAGUAGAACUUUGGUUUUCAACUUUUCCUACAGUGU>hsa-mir-4435-1 MI0016775AGGCAGCAAAUGGCCAGAGCUCACACAGAGGGAUGAGUGCACUUCACCUGCAGUGUGACUCAGCAGGCCAACAGAUGCUA>hsa-mir-4435-2 MI0016777GCAAAUGGCCAGAGCUCACACAGAGGGAUGAGUGCACUUCACCUGCAGUGUGACUCAGCAGGCCAACAGAUGCU>hsa-mir-4436a MI0016776GCCUCACUUUUCCACUUAUGCCUGCCCUGCCCCUCGAAUCUGCUCCACGAUUUGGGCAGGACAGGCAGAAGUGGAUAAGUGAGGA>hsa-mir-4436b-1 MI0017425GUGUCCUCACUUGUCCACUUCUGCCUGCCCUGCCCAAAUGGUGGAGCAGAUUCGAGGGGCAGGGCAGGAAGAAGUGGACAAGUGAGGCCAU>hsa-mir-4436b-2 MI0019110GUGUCCUCACUUGUCCACUUCUGCCUGCCCUGCCCAAAUGGUGGAGCAGAUUCGAGGGGCAGGGCAGGAAGAAGUGGACAAGUGAGGCCAU>hsa-mir-4437 MI0016778ACUUUGUGCAUUGGGUCCACAAGGAGGGGAUGACCCUUGUGGGCUCAGGGUACAAAGGUU>hsa-mir-4438 MI0016781UAAGUGUAAACUUAAGGACUGUCUUUUCUAAGCCUGUGCCUUGCCUUUCCUUUGGCACAGGCUUAGAAAAGACAGUCUUUAAGUUUACACUUC>hsa-mir-4439 MI0016782CCAGUGACUGAUACCUUGGAGGCAUUUUAUCUAAGAUACACACAAAGCAAAUGCCUCUAAGGUAUCAGUUUACCAGGCCA>hsa-mir-4440 MI0016783CUCUCACCAAGCAAGUGCAGUGGGGCUUGCUGGCUUGCACCGUGACUCCCUCUCACCAAGCAAGUGUCGUGGGGCUUGCUGGCUUGCACUGUGAAGAU>hsa-mir-4441 MI0016784CAGAGUCUCCUUCGUGUACAGGGAGGAGACUGUACGUGAGAGAUAGUCAGAUCCGCAUGUUAGAGCAGAGUCUCCUUCGUGUACAGGGAGGAGAUUGUAC>hsa-mir-4442 MI0016785GCGCCCUCCCUCUCUCCCCGGUGUGCAAAUGUGUGUGUGCGGUGUUAUGCCGGACAAGAGGGAGGUG>hsa-mir-4443 MI0016786GGUGGGGGUUGGAGGCGUGGGUUUUAGAACCUAUCCCUUUCUAGCCCUGAGCA>hsa-mir-4444-1 MI0016787GUGACGACUGGCCCCGCCUCUUCCUCUCGGUCCCAUAUUGAACUCGAGUUGGAAGAGGCGAGUCCGGUCUCAAA>hsa-mir-4444-2 MI0019111GUGACGACUGGCCCCGCCUCUUCCUCUCGGUCCCAUAUUGAACUCGAGUUGGAAGAGGCGAGUCCGGUCUCAAA>hsa-mir-4445 MI0016788UUCCUGCAGAUUGUUUCUUUUGCCGUGCAAGUUUAAGUUUUUGCACGGCAAAAGAAACAAUCCAGAGGGU>hsa-mir-4446 MI0016789CUGGUCCAUUUCCCUGCCAUUCCCUUGGCUUCAAUUUACUCCCAGGGCUGGCAGUGACAUGGGUCAA>hsa-mir-4447 MI0016790GUUCUAGAGCAUGGUUUCUCAUCAUUUGCACUACUGAUACUUGGGGUCAGAUAAUUGUUUGUGGUGGGGGCUGUUGUUUGCAUUGUAGGAU>hsa-mir-4448 MI0016791AGGAGUGACCAAAAGACAAGAGUGCGAGCCUUCUAUUAUGCCCAGACAGGGCCACCAGAGGGCUCCUUGGUCUAGGGGUAAUGCCA>hsa-mir-4449 MI0016792AGCAGCCCUCGGCGGCCCGGGGGGCGGGCGGCGGUGCCCGUCCCGGGGCUGCGCGAGGCACAGGCG>hsa-mir-4450 MI0016795UGUCUGGGGAUUUGGAGAAGUGGUGAGCGCAGGUCUUUGGCACCAUCUCCCCUGGUCCCUUGGCU>hsa-mir-4451 MI0016797UCUGUACCUCAGCUUUGCUCCCAACCAACCACUUCCACAUGUUUUGCUGGUAGAGCUGAGGACAGC>hsa-mir-4452 MI0016798UGGAUCACUUGAGGCCAAGAGUGCAAGGCUGUAGUGUGCACAGCCUUGAAUUCUUGGCCUUAAGUGAUCCC>hsa-mir-4453 MI0016799UGGAGAGCUUGGUCUGUAGCGGUUUCCUUCGGGGCAGGUGGGGACUGCUCCUUUGGGAGGAAGGAGGAGGCCCAGGCCGCGUCUUCAGG>hsa-mir-4454 MI0016800CCGGAUCCGAGUCACGGCACCAAAUUUCAUGCGUGUCCGUGUGAAGAGACCACCA>hsa-mir-4455 MI0016801AGAAGGGUGUGUGUGUUUUUCCUGAGAAUAAGAGAAGGAAGGACAGCCAAAUUCUUCA>hsa-mir-4456 MI0016802AUGAACCUGGUGGCUUCCUUUUCUGGGAGGAAGUUAGGGUUCA>hsa-mir-4457 MI0016803GGAGUACUCCAGUCAAUACCGUGUGAGUUAGAAAAGCUCAAUUCACAAGGUAUUGACUGGCGUAUUCA>hsa-mir-4458 MI0016804GAGCGCACAGAGGUAGGUGUGGAAGAAAGUGAAACACUAUUUUAGGUUUUAGUUACACUCUGCUGUGGUGUGCUG>hsa-mir-4459 MI0016805ACCCAGGAGGCGGAGGAGGUGGAGGUUGCAGUGAGCCAAGAUCGUGGCACUGACUCCAGCCUGGGG>hsa-mir-4460 MI0016806GUUUUUUGCCCAUAGUGGUUGUGAAUUUACCUUCUCCUCUUUGCAGUGAUAAAGGAGGUAAAUUCACAACCACUGUGGGCAGAAAC>hsa-mir-4461 MI0016807GAGUAGGCUUAGGUUAUGUACGUAGUCUAGGCCAUACGUGUUGGAGAUUGAGACUAGUAGGGCUAGGCCUACUG>hsa-mir-4462 MI0016810CUUCCCAGCUGCCCUAAGUCAGGAGUGGCUUUCCUGACACGGAGGGUGGCUUGGGAAA>hsa-mir-4463 MI0016811AAUAGAUUAUUGGUCACCACCUCCAGUUUCUGAAUUUGUGAGACUGGGGUGGGGCCUGAGAAUUUGC>hsa-mir-4464 MI0016812GGAACCUUAGUAAGGUUUGGAUAGAUGCAAUAAAGUAUGUCCACAGCUGAAAGGACAUACUUUAUUGCAUGUAUCCAAACCUUACUAAUUCA>hsa-mir-4465 MI0016816CAUGUGUCCCCUGGCACGCUAUUUGAGGUUUACUAUGGAACCUCAAGUAGUCUGACCAGGGGACACAUGA>hsa-mir-4466 MI0016817ACGCGGGUGCGGGCCGGCGGGGUAGAAGCCACCCGGCCCGGCCCGGCCCGGCGA>hsa-mir-4467 MI0016818UGGUGGCGGCGGUAGUUAUGGGCUUCUCUUUCUCACCAGCAGCCCCUGGGCCGCCGCCUCCCU>hsa-mir-4468 MI0016819AGUCUUCUCCUGGGGCUUUGGUGGCUAUGGUUGACUGGGCCACUCAGAGCAGAAGGAUGAGAUG>hsa-mir-4469 MI0016820CCGACGCGGAGAGCGGCUCUAGGUGGGUUUGGCGGCGGCGAGGACACCGCCGCUCCCUCUAGGGUCGCUCGGAGCGUGA>hsa-mir-4470 MI0016821CGAGCCUCUUUCGGCUUUCCAGUUUGUCUCGGUCCUUUGGAACGUGGCAAACGUGGAAGCCGAGAGGGCUCU>hsa-mir-4471 MI0016822CCAAAUUUAAAACUUAAACCUCUACUAAGUUUCCAUGAAAAGAACCCAUGGGAACUUAGUAGAGGUUUAAGUUUUAAAUUUGA>hsa-mir-4472-1 MI0016823UGGCAGACCCUUGCUCUCUCACUCUCCCUAAUGGGGCUGAAGACAGCUCAGGGGCAGGGUGGGGGGUGUUGUUUUUGUUU>hsa-mir-4472-2 MI0016824UGGUGGGGGUGGGGGGUGUUGUUUUUGUUUUUGAGACAGAGUCUUGCUCCGUCGCCCAGGCCGGAGU>hsa-mir-4473 MI0016825AAGGAACAGGGGACACUUGUAAUGGAGAACACUAAGCUAUGGACUGCUAUGGACUGCUAGUGCUCUCCGUUACAAGUAUCCCCUGUUACCU>hsa-mir-4474 MI0016826UUGCCUACCUUGUUAGUCUCAUGAUCAGACACAAAUAUGGCUCUUUGUGGCUGGUCAUGAGGCUAACAAGGUAGGCAC>hsa-mir-4475 MI0016827AUCUCAAUGAGUGUGUGGUUCUAAAUGACUCAUAGUCAAGGGACCAAGCAUUCAUUAUGAA>hsa-mir-4476 MI0016828AAAAGCCUGUCCCUAAGUCCCUCCCAGCCUUCCAGAGUUGGUGCCAGGAAGGAUUUAGGGACAGGCUUUG>hsa-mir-4477a MI0016829UCCUCCUCCCAUCAAUCACAAAUGUCCUUAAUGGCAUUUAAGGAUUGCUAUUAAGGACAUUUGUGAUUCACGGGAGGAGGU>hsa-mir-4477b MI0016830ACCUCCUCCCGUGAAUCACAAAUGUCCUUAAUAGCAAUCCUUAAAUGCCAUUAAGGACAUUUGUGAUUGAUGGGAGGAGGA>hsa-mir-4478 MI0016831GGCCGAGGCUGAGCUGAGGAGCCUCCAAACCUGUAGACAGGGUCAUGCAGUACUAGGGGCGAGCCUCAUCCCCUGCAGCCCUGGCC>hsa-mir-4479 MI0016838GAAACCAAGUCCGAGCGUGGCUGGCGCGGGAAAGUUCGGGAACGCGCGCGGCCGUGCUCGGAGCAGCGCCA>hsa-mir-4480 MI0016841GCAGAGGUGAGUUGACCUCCACAGGGCCACCCAGGGAGUAAGUAGCCAAGUGGAAGUUACUUUACCUCUGU>hsa-mir-4481 MI0016842GGAGUGGGCUGGUGGUUUUUUAAGAGGAAGGGAGACCUAAGCUAGCACAUGAGCACGCUC>hsa-mir-4482-1 MI0016843AGUGAGCAACCCAGUGGGCUA
UGGAAAUGUGUGGAAGAUGGCAUUUCUAUUUCUCAGUGGGGCUCUUACC>hsa-mir-4482-2 MI0017872AAUAAAAAUUGAAUAGUGAGCAACCCAGUGGGCUAUGGAAAUGUGUGGAAGAUGGCAUUUCUAUUUCUCAGUGGGGCUCUUACCUAUUACUCAUCAAC>hsa-mir-4483 MI0016844AAAAAACAACAUACUUAGUGCAUACCCAUAUAAUAUUAGGGGUGGUCUGUUGUUGUUUUUCU>hsa-mir-4484 MI0016845GGGUUUCCUCUGCCUUUUUUUCCAAUGAAAAUAACGAAACCUGUUAUUUCCCAUUGAGGGGGAAAAAGGCGGGAGAAGCCCCA>hsa-mir-4485 MI0016846AGAGGCACCGCCUGCCCAGUGACAUGCGUUUAACGGCCGCGGUACCCUAACUGUGCA>hsa-mir-4486 MI0016847GCAUGCUGGGCGAGGCUGGCAUCUAGCACAGGCGGUAGAUGCUUGCUCUUGCCAUUGCAAUGA>hsa-mir-4487 MI0016848ACUGUCCUUCAGCCAGAGCUGGCUGAAGGGCAGAAGGGAACUGUCCUUCAGCCAGAGCUGGCUGAAGGGCAGA>hsa-mir-4488 MI0016849GGUAGGGGGCGGGCUCCGGCGCUGGGACCCCACUAGGGUGGCGCCUUGGCCCCGCCCCGCCC>hsa-mir-4489 MI0016850GGGGGUGGGGCUAGUGAUGCAGGACGCUGGGGACUGGAGAAGUCCUGCCUGACCCUGUCCCA>hsa-mir-4490 MI0016852AUAGUUUCUGCAAUGCUCAAAUCUCUGGCCAAAGACCAGAACUUAAUGGUCUCUGGUAAGAGAUUUGGGCAUAUUAGAAACUAA>hsa-mir-4491 MI0016853ACAUUUGGUCACACCAGUCCACAUUAACGUGGACCAGACAAUAUUAAUGUGGACUGGUGUGACCAAAA>hsa-mir-4492 MI0016854CUGCAGCGUGCUUCUCCAGGCCCCGCGCGCGGACAGACACACGGACAAGUCCCGCCAGGGGCUGGGCGCGCGCCAGCCGG>hsa-mir-4493 MI0016855CCAGAGAUGGGAAGGCCUUCCGGUGAUUAUCACAGCCAUGCCUUUACCUCCAGAAGGCCUUUCCAUCUCUGUC>hsa-mir-4494 MI0016856AGUUUUAGUUACCCUGGUCAUCUGCAGUCUGAAAAUACAAAAUGGAAAAUUCCAGACUGUGGCUGACCAGAGGUAACUGAAACC>hsa-mir-4495 MI0016857AAGAAAUGUAAACAGGCUUUUUGCUCAGUGGAGUUAUUUUGAGCAAAAAGCUUAUUUACAUUUCUG>hsa-mir-4496 MI0016858ACAUCAGCUCAUAUAAUCCUCGAAGCUGCCUUUAGAAAUGAGGAAACUGAAGCUGAGAGGG>hsa-mir-4497 MI0016859ACCUCCGGGACGGCUGGGCGCCGGCGGCCGGGAGAUCCGCGCUUCCUGAAUCCCGGCCGGCCCGCCCGGCGCCCGUCCGCCCGCGGGUC>hsa-mir-4498 MI0016860AGGGCUGGGCUGGCAGGGCAAGUGCUGCAGAUCUUUGUCUAAGCAGCCCCUGCCUUGGAUCUCCCA>hsa-mir-4499 MI0016862AAGACUGAGAGGAGGGAACUGGUGAGUUGUACAUAGAAAUGCUUUCUAACUCCUUGUCUCAGUCUGUUU>hsa-mir-4500 MI0016863CAGGAGAGAAAGUACUGCCCAGAAGCUAAAGUGUAGAUCAAACGCAUAAUGGCUGAGGUAGUAGUUUCUUGAACUU>hsa-mir-4501 MI0016864UAUGUGACCUCGGAUGAAUCACUGAAAUAUGUCUGAGCUUCUGUUUCAUCAGAUGUCACAUUUU>hsa-mir-4502 MI0016865AGCCUUUAGCAAGUUGUAAUCUUUUUGCUGAUGGAGGGUCUUGCCUCCAUGGGGAUGGCUGAUGAUGAUGGUGCUGAAGGC>hsa-mir-4503 MI0016866ACAAUGUAGAUAUUUAAGCAGGAAAUAGAAUUUACAUAUAAAUUUCUAUUUGUUUCUAUUUCCUGCUUAAAUAUCUACAUUGC>hsa-mir-4504 MI0016867CUAAGAUAAUGUCCUCCAGGUUCAUCUCUGUUGUCAUUUGUGGCAUGGACCAUUUGUGACAAUAGAGAUGAACAUGGAGGAUAUUAUCUUAA>hsa-mir-4505 MI0016868GGAGGCUGGGCUGGGACGGACACCCGGCCUCCACUUUCUGUGGCAGGUACCUCCUCCAUGUCGGCCCGCCUUG>hsa-mir-4506 MI0016869UGGCCUCUGCCAUCAGACCAUCUGGGUUCAAGUUUGGCUCCAUCUUUAUGAAAUGGGUGGUCUGAGGCAAGUGGUCU>hsa-mir-4507 MI0016871UCUGGGCUGAGCCGAGCUGGGUUAAGCCGAGCUGGGUUGGGCUGGGCUGGGU>hsa-mir-4508 MI0016872AGGACCCAGCGGGGCUGGGCGCGCGGAGCAGCGCUGGGUGCAGCGCCUGCGCCGGCAGCUGCAAGGGCCG>hsa-mir-4509-1 MI0016873CUUUAAUACUAUCUCAAACUAAAGGAUAUAGAAGGUUUUCCCUUUCUCUUGCCCUGAAACCUUCUGUAUCCUUUAUUUUGAGAUAGUAUUAGAA>hsa-mir-4509-2 MI0016874CUUUAAUACUAUCUCAAACUAAAGGAUAUAGAAGGUUUUCCCUUUCUCUUGCCCUGAAACCUUCUGUAUCCUUUAUUUUGAGAUAGUAUUAGAA>hsa-mir-4509-3 MI0016875CUUUAAUACUAUCUCAAACUAAAGGAUAUAGAAGGUUUUCCCUUUCUCUUGCCCUGAAACCUUCUGUAUCCUUUAUUUUGAGAUAGUAUUAGAA>hsa-mir-4510 MI0016876GUGUAUGUGAGGGAGUAGGAUGUAUGGUUGUUAGAUAGACAACUACAAUCUUUUCUCACAACAGACAG>hsa-mir-4511 MI0016877AAAAAAAAGGGAAAGAAGAACUGUUGCAUUUGCCCUGCACUCAGUUUGCACAGGGUAAAUGCAAUAGUUCUUCUUUCCCUUUUUUUA>hsa-mir-4512 MI0016878CUCAGCCCGGGCAAUAUAGUGAGACCUCGUCUCUACAAAAAAUUGAGACAGGGCCUCACUGUAUCGCCCAGGCUGGA>hsa-mir-4513 MI0016879AUUCUAGGUGGGGAGACUGACGGCUGGAGGCCCAUAAGCUGUCUAAAACUUCGGCCCCCAGAUUUCUGGUCUCCCCACUUCAGAAC>hsa-mir-4514 MI0016880GUUGAGACAGGCAGGAUUGGGGAAACAUCUUUUACCUCGUCUCUUGCCUGUUUUAGA>hsa-mir-4515 MI0016881GCGGGAGGUGUAACAGGACUGGACUCCCGGCAGCCCCAGGGCAGGGGCGUGGGGAGCUGGUCCUAGCUCAGCGCUCCCGGA>hsa-mir-4516 MI0016882AGGGAGAAGGGUCGGGGCAGGGAGGGCAGGGCAGGCUCUGGGGUGGGGGGUCUGUGAGUCAGCCACGGCUCUGCCCACGUCUCCCC>hsa-mir-4517 MI0016883AGGUAAAUAUGAUGAAACUCACAGCUGAGGAGCUUAGCAAGUAGCUAAGGCCAGAGCUUGUGUUUGGGUGGUGUGGCUG>hsa-mir-4518 MI0016884UGGGGGAAAAGUGCUGGGAUUGAUUAGUGAUGUCUGCUGGGGAACCGGGGCUCAGGGAUGAUAACUGUGCUGAGAAGCCCCCU>hsa-mir-4519 MI0016885AACCUCAGCAGUGCGCAGGGCUGCACUGUCUCCGUCUGCGGCCUGCAGUAAGCGGGUA>hsa-mir-4520a MI0016886GUGUGCCACCUGCGUGUUUUCUGUCCAAAUCAGAAAAGGAUUUGGACAGAAAACACGCAGGAAGAAGGAA>hsa-mir-4520b MI0017358CCUGCGUGUUUUCUGUCCAAAUCCUUUUCUGAUUUGGACAGAAAACACGCAGGU>hsa-mir-4521 MI0016887UCGGCUAAGGAAGUCCUGUGCUCAGUUUUGUAGCAUCAAAACUAGGAUUUCUCUUGUUAC>hsa-mir-4522 MI0016889GCGGGCGUUGCCUGGGGGCCUCGCAGGGGGAGAUCCAGCCCAGGCUGGUUCCGCUGACUCUGCCUGUAGGCCGGUGGCGUCUUCUGG>hsa-mir-4523 MI0016890GCGGGGGACCGAGAGGGCCUCGGCUGUGUGAGGACUAGAGGCGGCCGAGGCCCGGGCCGGUUCCCCCGA>hsa-mir-4524a MI0016891GAACGAUAGCAGCAUGAACCUGUCUCACUGCAGAAUUAUUUUGAGACAGGCUUAUGCUGCUAUCCUUCA>hsa-mir-4524b MI0019114UAGCUGGGUGGAUGUGUUCUUUUGAAGGAUAGCAGCAUAAGCCUGUCUCAAAAUAAUUCUGCAGUGAGACAGGUUCAUGCUGCUAUCGUUCCAAAGAGGAAGGGUAAUCACUGUC>hsa-mir-4525 MI0016892GUCAGAGGGGGGAUGUGCAUGCUGGUUGGGGUGGGCUGCCUGUGGACCAAUCAGCGUGCACUUCCCCACCCUGAA>hsa-mir-4526 MI0016893UGCGGUGACAUCAGGGCCCAGUCCCUGCUGUCAUGCCCCAGGUGACGUGCUGGGCUGACAGCAGGGCUGGCCGCUAACGUCACUGUC>hsa-mir-4527 MI0016894CCAGAAGUGGUCUGCAAAGAGAUGACUGUGAAUCCAAGAUCCACAUCAGCUCUGUGCUGCCUACAUCUGA>hsa-mir-4528 MI0016895UAUUCUACUGAGAGUACAGAUCUUUAUAUAUAUGAUCAUUAUAUGUAUGAUGAGAUCAUUAUAUGUAUGAUCUGGACACCCAGUAGAAUC>hsa-mir-4529 MI0016896AUGACAGGCCAUCAGCAGUCCAAUGAAGACAUGAAGACCCAAUGUCUUCAUUGGACUGCUGAUGGCCCGUCACUGGGA>hsa-mir-4530 MI0016897CGACCGCACCCGCCCGAAGCUGGGUCAAGGAGCCCAGCAGGACGGGAGCGCGGCGC>hsa-mir-4531 MI0016898GCCUAGGAGUCCUUGGUCAGUGGGGACAUGGAGAAGGCUUCUGAGGA>hsa-mir-4532 MI0016899ACAGACCCCGGGGAGCCCGGCGGUGAAGCUCCUGGUAUCCUGGGUGUCUGA>hsa-mir-4533 MI0016900UGAGAAUGUGGAAGGAGGUUGCCGGACGCUGCUGGCUGCCUUCCAGCGUCCACUUCCCUUUCUCUCUCUCC>hsa-mir-4534 MI0016901UGUGAAUGACCCCCUUCCAGAGCCAAAAUCACCAGGGAUGGAGGAGGGGUCUUGGGUACU>hsa-mir-4535 MI0016903AACUGGGUCCCAGUCUUCACAGUUGGUUUCUGACACGUGGACCUGGCUGGGACGAUGUG>hsa-mir-4536-1 MI0016906AUGUGGUAGAUAUAUGCACGAUAUAUAUACUGCCCUGCUUUUAUACAUACAUACAUACAUACCUAUAUCGUGCAUAUAUCUACCACAU>hsa-mir-4536-2 MI0019149AUGUGGUAGAUAUAUGCACGAUAUAGGUAUGUAUGUAUGUAUGUAUAAAAGCAGGGCAGUAUAUAUAUCGUGCAUAUAUCUACCACAU>hsa-mir-4537 MI0016908UGAGCCGAGCUGAGCUUAGCUGGGCUGAGCUAACCAGGGCUGGGCUGAGCUGGGCUGAGCUGAGCUGAGC>hsa-mir-4538 MI0016909GAGCUUGGAUGAGCUGGGCUGAACUGGGCUGGGUUGAGCUGGGCUGGGCUGAGUUGAGCCAGGCUGAUCUGGGCUGAG>hsa-mir-4539 MI0016910UGAGCUGGGCUCUGCUGUGCUGUGCUGAGCAGGGCUGAGCUGAACUGGGCUGAGCUGGGC>hsa-mir-4540 MI0016911AAGCUGCAUGGACCAGGACUUGGCACCUUUGGCCUUAGUCCUGCCUGUAGGUUUA>hsa-mir-4632 MI0017259GAGGGCAGCGUGGGUGUGGCGGAGGCAGGCGUGACCGUUUGCCGCCCUCUCGCUGCUCUAG>hsa-mir-4633 MI0017260UGGCAAGUCUCCGCAUAUGCCUGGCUAGCUCCUCCACAAAUGCGUGUGGAGGAGCUAGCCAGGCAUAUGCAGAGCGUCA>hsa-mir-4634 MI0017261GGACAAGGGCGGCGCGACCGGCCCGGGGCUCUUGGGCGGCCGCGUUUCCCCUCC>hsa-mir-4635 MI0017262CCGGGACUUUGUGGGUUCUGACCCCACUUGGAUCACGCCGACAACACUGGUCUUGAAGUCAGAACCCGCAAAGUCCUGG>hsa-mir-4636 MI0017263UAGAUUCAGAACUCGUGUUCAAAGCCUUUAGCCCAGCAAUGGGAGAGUGCUAAAGGCUUCAAGCACGAGUUCUGAAUCUA>hsa-mir-4637 MI0017264CCCUUACUUGGAUCUGCAAUUAGUAUUUUAAUCAUAGAUUGUAUUUAGUUAGUUUUUAAUACUAACUGCAGAUUCAAGUGAGGG>hsa-mir-4638 MI0017265GACUCGGCUGCGGUGGACAAGUCCGGCUCCAGAACCUGGACACCGCUCAGCCGGCCGCGGCAGGGGUC>hsa-mir-4639 MI0017266UUGCUAAGUAGGCUGAGAUUGAUGUCAGGUUAUCCCCAAGCAUAACCUCACUCUCACCUUGCUUUGCAG>hsa-mir-4640 MI0017267CUGUGGGCUGGGCCAGGGAGCAGCUGGUGGGUGGGAAGUAAGAUCUGACCUGGACUCCAUCCCACCCACCCCCUGUUUCCUGGCCCACAG>hsa-mir-4641 MI0017268GGGGGGCAGGGGGCAGAGGGCAUCAGAGGACAGCCGCCUGGUGCCCAUGCCAUACUUUUGCCUCAG>hsa-mir-4642 MI0017269CACAACUGCAUGGCAUCGUCCCCUGGUGGCUGUGGCCUAGGGCAAGCCACAAAGCCACUCAGUGAUGAUGCCAGCAGUUGUG>hsa-mir-4643 MI0017270GUGUGCCCUAGCAUUUAUAAUCAUGUGUUCAUUCACAUGAUCAUAAGUGGACACAUGACCAUAAAUGCUAAAGCACAC>hsa-mir-4644 MI0017271GCGGCGGUGCUCUGCCUCUUUCUCCAUCCACCCUGGUCCAGGUCCACAGCAGUGGAGAGAGAAAAGAGACAGAAGGAUGGCCGU>hsa-mir-4645 MI0017272UGAUAGGGAAACCAGGCAAGAAAUAUUGUCUCCUCAAGUUGCGACGAGACAGUAGUUCUUGCCUGGUUUCUCUAUCA>hsa-mir-4646 MI0017273ACUGGGAAGAGGAGCUGAGGGACAUUGCGGAGAGGGUCUCACAUUGUCCCUCUCCCUUCCCAG>hsa-mir-4647 MI0017274CCAGGAGGGUGAAGAUGGUGCUGUGCUGAGGAAAGGGGAUGCAGAGCCCUGCCCAGCACCACCACCUCCUAUGCUCCUGG>hsa-mir-4648 MI0017275UGUGGGACUGCAAAUGGGAGCUCAGCACCUGCCUGCCACCCACGCAGACCAGCCCCUGCUCUGUUCCCACAG>hsa-mir-4649 MI0017276UCUGGGCGAGGGGUGGGCUCUCAGAGGGGCUGGCAGUACUGCUCUGAGGCCUGCCUCUCCCCAG>hsa-mir-4650-1 MI0017277UUCUGUAGAGAUUAUCAGGCCUCUUUCUACCUUCCAAGGCUCAGAAGGUAGAAUGAGGCCUGACAUAUCUGCAGGA>hsa-mir-4650-2 MI0017278UUCUGUAGAGAUUAUCAGGCCUCUUUCUACCUUCCAAGGCUCAGAAGGUAGAAUGAGGCCUGACAUAUCUGCAGGA>hsa-mir-4651 MI0017279CGGCGACGGCGGGGUGGGUGAGGUCGGGCCCCAAGACUCGGGGUUUGCCGGGCGCCUCAGUUCACCGCGGCCG>hsa-mir-4652 MI0017280UAUUGGACGAGGGGACUGGUUAAUAGAACUAACUAACCAGAACUAUUUUGUUCUGUUAACCCAUCCCCUCAUCUAAUA>hsa-mir-4653 MI0017281UUGUCCAAUUCUCUGAGCAAGGCUUAACACCAAAGGGUUAAGGGUUUGCUCUGGAGUUAAGGGUUGCUUGGAGAAUUGGAGAA>hsa-mir-4654 MI0017282CUGGCUGGUUGUGGGAUCUGGAGGCAUCUGGGGUUGGAAUGUGACCCCAGUCUCCUUUUCCCUCAUCAUCUGCCAG>hsa-mir-4655 MI0017283CCAAGGGCACACCGGGGAUGGCAGAGGGUCGUGGGAAAGUGUUGACCCUCGUCAGGUCCCCGGGGAGCCCCUGG>hsa-mir-4656 MI0017284AGGCUGGCGUGGGCUGAGGGCAGGAGGCCUGUGGCCGGUCCCAGGCCUCCUGCUUCCUGGGCUCAGGCUCGGUUU>hsa-mir-4657 MI0017285AAUGUGGAAGUGGUCUGAGGCAUAUAGAGUAUAUGCCAAGAACACUACCAUAU>hsa-mir-4658 MI0017286GCUGCCCUUCACUCAGAGCAUCUACACCCACUACCGGUGAGUGUGGAUCCUGGAGGAAUCGUGGC>hsa-mir-4659a MI0017287GAAACUGCUGAAGCUGCCAUGUCUAAGAAGAAAACUUUGGAGAAAAAUUUUCUUCUUAGACAUGGCAACGUCAACAGUUUC>hsa-mir-4659b MI0017291CUGUUGACGUUGCCAUGUCUAAGAAGAAAAUUUUUCUCCAAAGUUUUCUUCUUAGACAUGGCAGCUUCAGCAG>hsa-mir-4660 MI0017288ACUCCUUCUGCAGCUCUGGUGGAAAAUGGAGAAGACUUUUCCUUUCCUCCAUCUCCCCCAGGGCCUGGUGGAGU>hsa-mir-4661 MI0017289UUUACUCUGAACUAGCUCUGUGGAUCCUGACAGACAGCCUGAUAGACAGGAUCCACAGAGCUAGUCCAGAGUAAA>hsa-mir-4662a MI0017290UCUAUUUAGCCAAUUGUCCAUCUUUAGCUAUUCUGAAUGCCUAAAGAUAGACAAUUGGCUAAAUAGA>hsa-mir-4662b MI0017293CACAAUUUCUAUUUAGCCAAUUGUCUAUCUUUAGGCAUUCAGAAUAGCUAAAGAUGGACAAUUGGCUAAAUAGACACUGUG>hsa-mir-4663 MI0017292CUGUGGUGGAGCUGAGCUCCAUGGACGUGCAGUGGCAUCUGUCAUUGCUGCCUUCCUGGAGCUCAGGCCCUUGCAG>hsa-mir-4664 MI0017294GUUGGGGGCUGGGGUGCCCACUCCGCAAGUUAUCACUGAGCGACUUCCGGUCUGUGAGCCCCGUCCUCCGC>hsa-mir-4665 MI0017295CUCGAGGUGCUGGGGGACGCGUGAGCGCGAGCCGCUUCCUCACGGCUCGGCCGCGGCGCGUAGCCCCCGCCACAUCGGG>hsa-mir-4666a MI0017296AUCACUUAAAUACAUGUCAGAUUGUAUGCCUACAAAAUCCCUCCAGACUGGCAUACAAUC
UGACAUGUAUUUAAGAGAU>hsa-mir-4666b MI0019299UGUCUAAAUUGCAUGUCAGAUUGUAAUUCCCAGGCCCUUCCUCCAAUACUGGGAAUUACAAUUUGACAUGCAAUUUAGACA>hsa-mir-4667 MI0017297UGACUGGGGAGCAGAAGGAGAACCCAAGAAAAGCUGACUUGGAGGUCCCUCCUUCUGUCCCCACAG>hsa-mir-4668 MI0017298AGGGAAAAAAAAAAGGAUUUGUCUUGUAGCCAGGAUAUUGUUUUAAAGAAAAUCCUUUUUGUUUUUCCAG>hsa-mir-4669 MI0017300GCCUCCCUUCACUUCCUGGCCAUCCAGGCAUCUGUGUCUGUGUCCGGGAAGUGGAGGAGGGC>hsa-mir-4670 MI0017301CUCUAGGAAGCGACCAUGAUGUAACUUCACAGACUCUCCAAAAGUCUGAAGUUACAUCAUGGUCGCUUCCUAGAG>hsa-mir-4671 MI0017302UAUUUUAAGACCGAAGACUGUGCGCUAAUCUCUUAGCACUGAAGAUUAGUGCAUAGUCUUUGGUCUCAAAAUA>hsa-mir-4672 MI0017303GGCUGCUUCUCGCCUCUGUCCAGCUGUGUGGCCUUGGACAAGCCUCUUGGUUACACAGCUGGACAGAGGCACGAAACAGCC>hsa-mir-4673 MI0017304GUCCAGGCAGGAGCCGGACUGGACCUCAGGGAAGAGGCUGACCCGGCCCCUCUUGCGGC>hsa-mir-4674 MI0017305CCCAGGCGCCCGCUCCCGACCCACGCCGCGCCGCCGGGUCCCUCCUCCCCGGAGAGGCUGGGCUCGGGACGCGCGGCUCAGCUCGGG>hsa-mir-4675 MI0017306CAUGAGAAAUCCUGCUGGUCAACCAUAGCCCUGGUCAGACUCUCCGGGGCUGUGAUUGACCAGCAGGACUUCUCAUG>hsa-mir-4676 MI0017307UGAAUGAAAGAGCCAGUGGUGAGACAGUGAGUUGAUUACUUCUCACUGUUUCACCACUGGCUCUUUGGUUCA>hsa-mir-4677 MI0017308GCAAAGCAGCAAUUGUUCUUUGGUCUUUCAGCCAUGACCUGACCUUCUGUCUGUGAGACCAAAGAACUACUUUGCUUGGC>hsa-mir-4678 MI0017309GGAAAAAACAAGGUAUUGUUCAGACUUAUGAUUUUUGGGGUCAAAGAUUCUGAGCAAUAACCUAUUAAAAAACC>hsa-mir-4679-1 MI0017310GUCUUUUUUCUGUGAUAGAGAUUCUUUGCUUUGUUAGAAACAAAAAGCAAAGAAUCUCUAUCACAGAAAAAAGAU>hsa-mir-4679-2 MI0017311UAUCUUUUUUCUGUGAUAGAGAUUCUUUGCUUUUUGUUUCUAACAAAGCAAAGAAUCUCUAUCACAGAAAAAAGACG>hsa-mir-4680 MI0017312UAUAAGAACUCUUGCAGUCUUAGAUGUUAUAAAAAUAUAUAUCUGAAUUGUAAGAGUUGUUAGCAC>hsa-mir-4681 MI0017313GGCAACGGGAAUGCAGGCUGUAUCUGCAGGGCAUUGUGCUAACAGGUGCAGGCUGCAGACCUGUCACAGGCC>hsa-mir-4682 MI0017314UGCCCCUGGUCUGAGUUCCUGGAGCCUGGUCUGUCACUGGGGAAGUCCAGAGCUCCAAGGCUCAGUGCCCAGGGGACGCA>hsa-mir-4683 MI0017315GACACGCAAGACGAGGCGGGCCUGGAGGUGCACCAGUUCUGGCCGCUGGUGGAGAUCCAGUGCUCGCCCGAUCUCAAGUUC>hsa-mir-4684 MI0017316GCACCAGGGGUACCUCUCUACUGACUUGCAACAUACAUUUGUCUUGGUGUGUUGCAAGUCGGUGGAGACGUACCCUUGGUGC>hsa-mir-4685 MI0017317UAGCCCAGGGCUUGGAGUGGGGCAAGGUUGUUGGUGAUAUGGCUUCCUCUCCCUUCCUGCCCUGGCUAG>hsa-mir-4686 MI0017318GGCUUCCUGUAUCUGCUGGGCUUUCUGGUGUUGGCAGCCCAAGAUGACACCCUGGGCCCAGCAGGAGCCAGAAGCC>hsa-mir-4687 MI0017319ACCUGAGGAGCCAGCCCUCCUCCCGCACCCAAACUUGGAGCACUUGACCUUUGGCUGUUGGAGGGGGCAGGCUCGCGGGU>hsa-mir-4688 MI0017321GUCUACUCCCAGGGUGCCAAGCUGUUUCGUGUUCCCUCCCUAGGGGAUCCCAGGUAGGGGCAGCAGAGGACCUGGGCCUGGAC>hsa-mir-4689 MI0017322GGUUUCUCCUUGAGGAGACAUGGUGGGGGCCGGUCAGGCAGCCCAUGCCAUGUGUCCUCAUGGAGAGGCC>hsa-mir-4690 MI0017323GAGCAGGCGAGGCUGGGCUGAACCCGUGGGUGAGGAGUGCAGCCCAGCUGAGGCCUCUGC>hsa-mir-4691 MI0017324GGAGCACUCCCAGGUCCUCCAGGCCAUGAGCUGCGGCCCUGAUGUCUCUACUCCAGCCACGGACUGAGAGUGCAUAGGAGUGUCC>hsa-mir-4692 MI0017325GUUUUACUUGAUACCCACACUGCCUGGGUGGGACACUCAGGCAGUGUGGGUAUCAGAUAAAAC>hsa-mir-4693 MI0017326GUUUAAAGAAUACUGUGAAUUUCACUGUCACAAAUUCAAAUAAAGUGAGAGUGGAAUUCACAGUAUUUAAGGAAU>hsa-mir-4694 MI0017327CAAAUACAUAGGUGUUAUCCUAUCCAUUUGCCUCUCUCAGAAAAUAGAGUCAAAUGGACAGGAUAACACCUAUGUAUUUG>hsa-mir-4695 MI0017328CCUGCAGGAGGCAGUGGGCGAGCAGGCGGGGCAGCCCAAUGCCAUGGGCCUGAUCUCACCGCUGCCUCCUUCCC>hsa-mir-4696 MI0017329CAAAGCCACUGCAAGACGGAUACUGUCAUCUAUUCCAGAAGAUGACAAUGUCCAUUUUGCAGUGGCUUUG>hsa-mir-4697 MI0017330GGGCCCAGAAGGGGGCGCAGUCACUGACGUGAAGGGACCACAUCCCGCUUCAUGUCAGUGACUCCUGCCCCUUGGUCU>hsa-mir-4698 MI0017331UGCUUCUCCUGGGGUCUUCCUCUACAUUUCCACCUAGACGGGCCUGGGUCAAAAUGUAGAGGAAGACCCCAGAAGGAGCA>hsa-mir-4699 MI0017332AGCAAUUGGAGAAGAUUGCAGAGUAAGUUCCUGAUUAAGAAAUGGAAUUUACUCUGCAAUCUUCUCCAAUUGCU>hsa-mir-4700 MI0017333UCAGUGAGGUCUGGGGAUGAGGACAGUGUGUCCUGAAAUUCACAGGACUGACUCCUCACCCCAGUGCACGAGGA>hsa-mir-4701 MI0017334CCUUGGCCACCACACCUACCCCUUGUGAAUGUCGGGCAAUGGGUGAUGGGUGUGGUGUCCACA>hsa-mir-4703 MI0017336UUAUGCAUAUUAGCAAUACAGUACAAAUAUAGUGUGUUUGAUUUGCACUGUAGUUGUAUUGUAUUGCCACUCUGUAUAA>hsa-mir-4704 MI0017337CUUAUCCUAGACACUAGGCAUGUGAGUGAUUGUCUUCCUCACUCAAUCAGUCACAUAUCUAGUGUCUAGAAUGAG>hsa-mir-4705 MI0017338CUCACAAGAUCAAUCACUUGGUAAUUGCUGUGAUAACAACUCAGCAAUUACCAAGUGAUUGGUUUUGUGAG>hsa-mir-4706 MI0017339GCUACGGGGAGCGGGGAGGAAGUGGGCGCUGCUUCUGCGUUAUCUGGAAGGAGCAGCCCACUCCUGUCCUGGGCUCUGUGGU>hsa-mir-4707 MI0017340GGUUCCGGAGCCCCGGCGCGGGCGGGUUCUGGGGUGUAGACGCUGCUGGCCAGCCCGCCCCAGCCGAGGUUCUCGGCACC>hsa-mir-4708 MI0017341UUUAGGAGAGAGAUGCCGCCUUGCUCCUUGAACAGGAGGAGCAAGGCGGCAUCUCUCUGAUACUAAA>hsa-mir-4709 MI0017342CUGCUUCAACAACAGUGACUUGCUCUCCAAUGGUAUCCAGUGAUUCGUUGAAGAGGAGGUGCUCUGUAGCAG>hsa-mir-4710 MI0017344GACCGAGUGGGGUGAGGGCAGGUGGUUCUUCCCGAAGCAGCUCUCGCCUCUUCGUC>hsa-mir-4711 MI0017345AAAUGUGCAUCAGGCCAGAAGACAUGAGCCCUUUGGAAAGGUCUCGUGUCUUCUGGCUUGAUGCACAUUU>hsa-mir-4712 MI0017346GACAGGAUUCCAGUACAGGUCUCUCAUUUCCUUCAUGAUUAGGAAUACUACUUUGAAAUGAGAGACCUGUACUGUAUCUGUU>hsa-mir-4713 MI0017347GUCCCCAUUUUUCUCCCACUACCAGGCUCCCAUAAGGGUCGAAUGGGAUCCAGACAGUGGGAGAAAAAUGGGGAC>hsa-mir-4714 MI0017348AUUUUGGCCAACUCUGACCCCUUAGGUUGAUGUCAGAAUGAGGUGUACCAACCUAGGUGGUCAGAGUUGGCCAAAAU>hsa-mir-4715 MI0017349GGGGAAUGAAAGUUGGCUGCAGUUAAGGUGGCUAAUCAGCUGAUGGUGCCACCUUAACUGCAGCCAAUUCUAAUUCCCC>hsa-mir-4716 MI0017350CAUACUUUGUCUCCAUGUUUCCUUCCCCCUUCUGUAUACAUGUAUACAGGAGGAAGGGGGAAGGAAACAUGGAGACAAAGUGUG>hsa-mir-4717 MI0017352GGCAGUGUUUAGGCCACAGCCACCCAUGUGUAGGGGUGGCUACACAUGGGUGGCUGUGGCCUAAACACUGCC>hsa-mir-4718 MI0017353AGCUGUACCUGAAACCAAGCACCUGUUUGUGACUUGGCUUCAGUUACUAGC>hsa-mir-4719 MI0017354ACAAUGAUGACUUGUAUGUUAUAGAUUUGUGAUUACAUUAAAACUUAAAAUUUCACAAAUCUAUAAUAUGCAGGUCAUCACUGU>hsa-mir-4720 MI0017355AAGCCUGGCAUAUUUGGUAUAACUUAAGCACCAGGUAAAAUCUGGUGCUUAAGUUGUACCAAGUAUAGCCAAGUUU>hsa-mir-4721 MI0017356GGGCCUGGUCAUGGUCAAGCCAGGUUCCAUCAAGCCCCACCAGAAGGUGGAGGCCCAGGUGAGGGCUCCAGGUGACGGUGGGCAGGGUU>hsa-mir-4722 MI0017357GGCAGGAGGGCUGUGCCAGGUUGGCUGGGCCAGGCCUGACCUGCCAGCACCUCCCUGCAG>hsa-mir-4723 MI0017359AGUUGGUGGGGGAGCCAUGAGAUAAGAGCACCUCCUAGAGAAUGUUGAACUAAAGGUGCCCUCUCUGGCUCCUCCCCAAAG>hsa-mir-4724 MI0017361ACGCAAAAUGAACUGAACCAGGAGUGAGCUUCGUGUACAUUAUCUAUUAGAAAAUGAAGUACCUUCUGGUUCAGCUAGUCCCUGUGCGU>hsa-mir-4725 MI0017362GUGUCUCUCUGGAGACCCUGCAGCCUUCCCACCCACCAGGGAGCUUUCCAUGGGCUGUGGGGAAGGCGUCAGUGUCGGGUGAGGGAACAC>hsa-mir-4726 MI0017363AGGGCCAGAGGAGCCUGGAGUGGUCGGGUCGACUGAACCCAGGUUCCCUCUGGCCGCA>hsa-mir-4727 MI0017364AAUCUGCCAGCUUCCACAGUGGCAGAUUUUCCCAUAGUGGGAAGCUGGCAGAUUC>hsa-mir-4728 MI0017365GUGGGAGGGGAGAGGCAGCAAGCACACAGGGCCUGGGACUAGCAUGCUGACCUCCCUCCUGCCCCAG>hsa-mir-4729 MI0017366UCUGUUUCCUCAUUUAUCUGUUGGGAAGCUAACUGUGACCUUAGCGUCCCAGCAGAUAAAUGAGGAAACAGA>hsa-mir-4730 MI0017367CGCAGGCCUCUGGCGGAGCCCAUUCCAUGCCAGAUGCUGAGCGAUGGCUGGUGUGUGCUGCUCCACAGGCCUGGUG>hsa-mir-4731 MI0017368CCCUGCCAGUGCUGGGGGCCACAUGAGUGUGCAGUCAUCCACACACAAGUGGCCCCCAACACUGGCAGGG>hsa-mir-4732 MI0017369GAGGGAGCUGUAGAGCAGGGAGCAGGAAGCUGUGUGUGUCCAGCCCUGACCUGUCCUGUUCUGCCCCCAGCCCCUC>hsa-mir-4733 MI0017370GGUCGCUUAAAUCCCAAUGCUAGACCCGGUGGCAAUCAAGGUCUAGCCACCAGGUCUAGCAUUGGGAUUUAAGCCC>hsa-mir-4734 MI0017371CUCGGGCCCGACCGCGCCGGCCCGCACCUCCCGGCCCGGAGCUGCGGGCUGCGGUCAGGGCGAUCCCGGG>hsa-mir-4735 MI0017372UGCAGUGCCUAAUUUGAACACCUUCGGUAUUCAUCAAAAAUACCAAAGGUGCUCAAAUUAGACAUUGCA>hsa-mir-4736 MI0017373AGGCAGGUUAUCUGGGCUGCCAUCUCCCACUGGCUGCUUGCCUGCCU>hsa-mir-4737 MI0017374CUGCACAGGAUGCGAGGAUGCUGACAGUGCCUCACAGCCGCACAGGACCGAGGAUGCUGACGGUGCCUCACAGCCACACAG>hsa-mir-4738 MI0017376GGUCGCAUUUCUCCUUCUUACCAGCGCGUUUUCAGUUUCAUAGGGAAGCCUUUCCAUGAAACUGGAGCGCCUGGAGGAGAAGGGGCC>hsa-mir-4739 MI0017377GGGAGGAAGAAGGGAGGAGGAGCGGAGGGGCCCUUGUCUUCCCAGAGCCUCUCCCUUCCUCCCCUCCCCCUCCC>hsa-mir-4740 MI0017378GCCAAGGACUGAUCCUCUCGGGCAGGGAGUCAGAGGGGACCGCCCGAGAGGAUCCGUCCCUGC>hsa-mir-4741 MI0017379CGGGCGGGGCGGGUCCGGCCGCCUCCGAGCCCGGCCGGCAGCCCCCGGCCUUAAAGCGCGGGCUGUCCGGAGGGGUCGGCUUUCCCACCG>hsa-mir-4742 MI0017380UCAGGCAAAGGGAUAUUUACAGAUACUUUUUAAAAUUUGUUUGAGUUGAGGCAGAUUAAAUAUCUGUAUUCUCCUUUGCCUGCAG>hsa-mir-4743 MI0017381GCUGGCCGGAUGGGACAGGAGGCAUGAAUGAGCCAUCUUUCCAAUGCCUUUCUGUCUUUUCUGGUCCAG>hsa-mir-4744 MI0017382GUAAUCACAUCUAAAGACUAGACUUCGCUAUGACCAGGCCAUAGUAAACAUCAUAGUAUGUCUAGUCUUUAGGUUUGAUUAC>hsa-mir-4745 MI0017384GUGAGUGGGGCUCCCGGGACGGCGCCCGCCCUGGCCCUGGCCCGGCGACGUCUCACGGUCCC>hsa-mir-4746 MI0017385GUGUCUGUGCCGGUCCCAGGAGAACCUGCAGAGGCAUCGGGUCAGCGGUGCUCCUGCGGGCCGACACUCAC>hsa-mir-4747 MI0017386AGGGAAGGAGGCUUGGUCUUAGCACGGGGUCUAAGGCCCGGGCUUUCCUCCCAG>hsa-mir-4748 MI0017387UGGCUGGCUGAGGUUUGGGGAGGAUUUGCUGGUGCUAGAGAGGAAAGCAGACCCUACCCAACCCCACGCCCUACUACAGCCA>hsa-mir-4749 MI0017388CCUGCGGGGACAGGCCAGGGCAUCUAGGCUGUGCACAGUGACGCCCCUCCUGCCCCCACAG>hsa-mir-4750 MI0017389CGCUCGGGCGGAGGUGGUUGAGUGCCGACUGGCGCCUGACCCACCCCCUCCCGCAG>hsa-mir-4751 MI0017390CCCGGAGCCAGAGGACCCGUAGCUGCUAGAAGGGCAGGGGUGUGGCUUCUGGGGGCUGGUCUUCAGCUCUGGCG>hsa-mir-4752 MI0017391AGUGUCUCCUUGUGGAUCUCAAGGAUGUGCUUCCACAUAGCAGCAUGUUCUUCAGAUGGACAAGGAGACACU>hsa-mir-4753 MI0017392AUAUCUACACAAGGCCAAAGGAAGAGAACAGAUAUAUCCACAGUACACUUGGCUGUUCUCUUUCUUUAGCCUUGUGUAGAUAU>hsa-mir-4754 MI0017394ACGCGCCUGAUGCGGACCUGGGUUAGCGGAGUGAGGCCCAGUGGUCACCGCCGCCCUCCGCAGGUCCAGGUUGCCGUGCGCAUGUGCCU>hsa-mir-4755 MI0017395AGAUUCAGCUUUCCCUUCAGAGCCUGGCUUUGGCAUCUAUGAAAGCCAGGCUCUGAAGGGAAAGUUGAAUCU>hsa-mir-4756 MI0017397GGGAUAAAAUGCAGGGAGGCGCUCACUCUCUGCUGCCGAUUCUGCACCAGAGAUGGUUGCCUUCCUAUAUUUUGUGUC>hsa-mir-4757 MI0017398UUCCAGCCCGAGGCCUCUGUGACGUCACGGUGUCUGCGGGAGGAGACCAUGACGUCACAGAGGCUUCGCGCUCUGAG>hsa-mir-4758 MI0017399GGUGAGUGGGAGCCGGUGGGGCUGGAGUAAGGGCACGCCCGGGGCUGCCCCACCUGCUGACCACCCUCCCC>hsa-mir-4759 MI0017400CAUUUAGGACUAGAUGUUGGAAUUAGACAGAAAAAAGUUAGACACAAAAAAUUGUGUCUAAUUCCAACAUCUAGUCCUAAAUG>hsa-mir-4760 MI0017401GCCAUGGUGUUUAGAUUGAACAUGAAGUUAGAAUUCUUAAGUAUCAAAACUAAAUUCAUGUUCAAUCUAAACCCCAUGGC>hsa-mir-4761 MI0017402GGACAAGGUGUGCAUGCCUGACCCGUUGUCAGACCUGGAAAAAGGGCCGGCUGUGGGCAGGGAGGGCAUGCGCACUUUGUCC>hsa-mir-4762 MI0017403CUGAUACCCCAAAUCUUGAUCAGAAGCCUUGAUCAGAAGCUAGGAAGGCUUCUGAUCAAGAUUUGUGGUGUCAAG>hsa-mir-4763 MI0017404CCUGUCCCUCCUGCCCUGCGCCUGCCCAGCCCUCCUGCUCUGGUGACUGAGGACCGCCAGGCAGGGGCUGGUGCUGGGCGGGGGGCGGCGGG>hsa-mir-4764 MI0017405UCUUCCCCAUGGAUGUGGAAGGAGUUAUCUGUCACCAGUCAGAUAACUGUCACCAGUCAGUUAACUCCUUUCACACCCAUGGGGAAGA>hsa-mir-4765 MI0017406UGGUGAUUUUGAACGUAGCUAUCCACCACUCAGCCUGGAAAAAGCUGAGUGAUUGAUAGCUAUGUUCAAAAUCACCA>hsa-mir-4766 MI0017407CUGAAGCUCCUUCUGAAAGAGCAGUUGGUGUUUAUUUUUUACUAAAUAGCAAUUGCUCUUUUGGAAGGAACUUGAG>hsa-mir-4767 MI0017408ACAUGGGCCCGCGGGCGCUCCUGGCCGCCGCCCGACUUCGGGGCCAGCCGGGGGCAGA
GCGCGCGGGAGCCCGAGCGU>hsa-mir-4768 MI0017409AAACUUUGAUUCUCUCUGGAUCCCAUGGAUAUGGGAACUGUGAUGUCCAGGAGAUCCAGAGAGAAUCAGAGUUU>hsa-mir-4769 MI0017410GAGGAGAGGUGGGAUGGAGAGAAGGUAUGAGCUAAAAAUCCCCAAGCUCUGCCAUCCUCCCUCCCCUACUUCUCCCC>hsa-mir-4770 MI0017411GAGUUAUGGGGUCAUCUAUCCUUCCCUUGGAAAAUGAUCUGAGAUGACACUGUAGCUC>hsa-mir-4771-1 MI0017412GCUCUAGCCUAAUUUUAGAUCUGGUCUGCUUCAGUUUCACUCCAAGCAGACUUGACCUACAAUUAGCCUAGAGC>hsa-mir-4771-2 MI0017413GCUCUAGCCUAAUUUUAGAUCUGGUCUGCUUCAGUUUCACUCCAAGCAGACUUGACCUACAAUUAGCCUAGAGC>hsa-mir-4772 MI0017414GUGAUUGCCUCUGAUCAGGCAAAAUUGCAGACUGUCUUCCCAAAUAGCCUGCAACUUUGCCUGAUCAGAGGCAGUCAC>hsa-mir-4773-1 MI0017415UGCUCCCCAGCCUUUCUAUGCUCCUGUUCUGCUUUAUUUCAUCAAAGCAGAACAGGAGCAUAGAAAGGCUGGGGAGCA>hsa-mir-4773-2 MI0017416UGCUCCCCAGCCUUUCUAUGCUCCUGUUCUGCUUUGAUGAAAUAAAGCAGAACAGGAGCAUAGAAAGGCUGGGGAGCA>hsa-mir-4774 MI0017417UAUAUUGUUGUCUGGUAUGUAGUAGGUAAUAACUGACAAACAGACAAUUGCCUAACAUGUGCCAGAAAACAACAUA>hsa-mir-4775 MI0017418AUUAAGCUUUUAAUUUUUUGUUUCGGUCACUCUUGAUAGCAGACAUUGACUGAAACAAAAAAUUAAAAGCUUUAU>hsa-mir-4776-1 MI0017419CUAUAUGCAGUGGACCAGGAUGGCAAGGGCUCUCCUGAAAGGACAGUAGAGCCCUUGCCAUCCUGGUCCACUGCAUAUAG>hsa-mir-4776-2 MI0017420CUAUAUGCAGUGGACCAGGAUGGCAAGGGCUCUACUGUCCUUUCAGGAGAGCCCUUGCCAUCCUGGUCCACUGCAUAUAG>hsa-mir-4777 MI0017421UAGAAUAUUUCGGCAUUCUAGAUGAGAGAUAUAUAUAUACCUCAUAUGUAUAUGGUAUACCUCAUCUAGAAUGCUGUAAUAUUCUA>hsa-mir-4778 MI0017422UCACAUGUCCAAUUCUGUAAAGGAAGAAGAGGUAAGAAGAAGUGAAGCCCUCUUCUUCCUUUGCAGAGUUGAAUAUGUGG>hsa-mir-4779 MI0017423UAAAUGUCUUACUGCUUUUACUGUUCCCUCCUAGAGUCCAUUCUUUACUCUAGGAGGGAAUAGUAAAAGCAGUAAGACAUUUA>hsa-mir-4780 MI0017424GGCCAGUGCCAGGGGGUCAGGCUCAAGGACCAGCCCAAAGGCCAGGCCUGACCCUUGAGCCUGAUCCCUAGCACUGAUCCC>hsa-mir-4781 MI0017426AGGUGCACGCUCUAGCGGGGAUUCCAAUAUUGGGCCAAUUCCCCCAAUGUUGGAAUCCUCGCUAGAGCGUGCACUU>hsa-mir-4782 MI0017427AUUGCCCAGUUCUGGAUAUGAAGACAAUCAAGAAAAGAUUUGGUGUUCUUGAUUGUCUUCAUAUCUAGAACUGGGCAGU>hsa-mir-4783 MI0017428GGGAAAGCGGAGGGCGCGCCCAGCUCCCGGGCUGAUUGCGCUAACAGUGGCCCCGGUGUUGGGGCGCGUCUGCCGCUGCCCC>hsa-mir-4784 MI0017429UGACUGGGCUGAGGAGAUGCUGGGACUGAGAGUGUCAUGGUGGAGCCUCCGUCCCUGCUCAUCCUCUCCGCAUGUUG>hsa-mir-4785 MI0017430GUAGGUGGGGACGCGGCGGCGCUGCUCCUCCGCUGCCGCCGGGAGAGUCGGCGACGCCGCCAGCUCCGCGCGC>hsa-mir-4786 MI0017433GGGCAUGGCCUGAGACCAGGACUGGAUGCACCACUCUCCCUGUGAUGAGGUGAAGCCAGCUCUGGUCUGGGCCAUUUCAC>hsa-mir-4787 MI0017434CGGUCCAGACGUGGCGGGGGUGGCGGCGGCAUCCCGGACGGCCUGUGAGGGAUGCGCCGCCCACUGCCCCGCGCCGCCUGACCG>hsa-mir-4788 MI0017435AAUGAAGGAUUACGGACCAGCUAAGGGAGGCAUUAGGAUCCUUAUUCUUGCCUCCCUUAGUUGGUCCCUAAUCCUUCGUU>hsa-mir-4789 MI0017436CAUGCUACGUAUGUAUACACCUGAUAUGUGUAUGUGUAAAUACAUAUCCACACACAUAGCAGGUGUAUAUAUAGGUAGCCUG>hsa-mir-4790 MI0017437CAAUGUGACAUCGCUUUACCAUUCAUGUUCACUGAAAGGUAGAUUUUAAAAACAUGAAUGGUAAAGCGAUGUCACAUUG>hsa-mir-4791 MI0017438UAAGAACUGGAUAUGAUGACUGAAAUAAGCUCCAUAUCAAUGAGAAUUUCAAUGGGAUUAUGUGCAGUCAAUGUCCAGUAAUUA>hsa-mir-4792 MI0017439GCAGCCCGGUGAGCGCUCGCUGGCCUGGCAGUGCGUCGGAAGAACAGGGCGGGUGGGGCCGCGCACAUCUCUGC>hsa-mir-4793 MI0017440UUUCUCCUCGCUGCCCGCACAUCCUGCUCCACAGGGCAGAGGGAGGCCAAGAAGACCUCUGCACUGUGAGUUGGCUGGCUGGAGGAA>hsa-mir-4794 MI0017441UUUUAACAUCUGGCUAUCUCACGAGACUGUAUGUCCUAACAGUGCUUGUAGUCUCAUGAGAUAGCCAGAUGUUAAAA>hsa-mir-4795 MI0017442UGAUAUGGAAGAAAUCCAGAAGUGGCUAAUAAUAUUGACACUAUAACAAUAAUGUCAAUAUUAUUAGCCACUUCUGGAUUUAUGAAUCA>hsa-mir-4796 MI0017443UAAAUUUGUGUCUAUACUCUGUCACUUUACUUUUGGCCUCAAGUCAUUGCAGUAAAGUGGCAGAGUAUAGACACAAAUUUA>hsa-mir-4797 MI0017444GACUCAGAAGACAGAGUGCCACUUACUGAAAGGUUUUUUCUCUCAGUAAGUGGCACUCUGUCUUCUGAGUU>hsa-mir-4798 MI0017445AAGUACAACUUCGGUAUACUUUGUGAAUUGGCUUUUACAAAAGACCAACUCACGAAGUAUACCGAAGUCAUACUU>hsa-mir-4799 MI0017446ACUGCUAAUAUCUAAAUGCAGCAUGCCAGUCCUGAGAUGCAGGGACUGGCAUGCUGCAUUUAUAUAUUAGCAGU>hsa-mir-4800 MI0017448GGAGAAAGGAGUGGACCGAGGAAGGAAGGAAGGCAAGGCUGUCUGUCCAUCCGUCCGUCUGUCCACCUACCUGUCAGUCC>hsa-mir-4801 MI0017449UUGAGGCUUGGUUUUCUUAUGUGUAAAAUGUAAUAACAUUUCUUAUGUUUAAAACACUUUACACAAGAAAACCAAGGCUCAA>hsa-mir-4802 MI0017450CUGACUGGCUUGUAUGGAGGUUCUAGACCAUGUUAGUGUUCAAGUCUACAUGGAUGGAAACCUUCAAGCAGGCCAAGCAG>hsa-mir-4803 MI0017451AGUGGGAUUUAACAUAAUAGUGUGGAUUGAAUCACACACACAUUUCAACCCACACUAUGAUGUUAAAUCCCAUU>hsa-mir-4804 MI0017452UCAGUGUAUUUGGACGGUAAGGUUAAGCAAGGUGCGUCGUAUCUUGCUUAACCUUGCCCUCGAAAUACACUGA>hsa-mir-4999 MI0017865AUAGAAAAUAAAACACAUACUGCUGUAUUGUCAGGUAGUGAUAGGAUUUAUCACUACCUGACAAUACAGUAUGUGUUUGUUUUAUAUAUUU>hsa-mir-5000 MI0017866CUGAAGAGUAGAGUGUGUGGUCCCAGUUCAGAAGUGUUCCUGAGUAACUUGUGCUUAUAACUCAGGACACUUCUGAACUUGGACCAUACAGGUCUCCCUGCUU>hsa-mir-5001 MI0017867AGCUCAGGGCGGCUGCGCAGAGGGCUGGACUCAGCGGCGGAGCUGGCUGCUGGCCUCAGUUCUGCCUCUGUCCAGGUCCUUGUGACCCGCCCGCUCUCCU>hsa-mir-5002 MI0017868UCUUCCUCUCUGUCCUCUGGAAUUUGGUUUCUGAGGCACUUAGUAGGUGAUAGCAUGACUGACUGCCUCACUGACCACUUCCAGAUGAGGGUUACUC>hsa-mir-5003 MI0017869AUGAGUUUGCUUUGUGUCAUCCUCACAACAACCUUGCAGGGUAGAGAUGAUUUUUCCUACUUUUCUAGGUUGUUGGGGGCUGGGGCAGGGGGAACAGAG>hsa-mir-5004 MI0017870GGCACUUGCUUGGGGGUUAGUGAGGACAGGGCAAAUUCACGAGAUUGGGUUGUGCAGAGGCUGACACUUGGAUUUUCCUGGGCCUCAGGACUUCCUUUCAGACAUGG>hsa-mir-5006 MI0017873AACCAUUAGGGGGCUGUGGUUUGCCAGGGCAGGAGGUGGAAGGGAGCCCCAUUUACAGUGGUAACUUCCUUUCCCUUUCCAUCCUGGCAGGCUUCAGAGAACUUUACCAG>hsa-mir-5007 MI0017874GGUAAACCUUGGUGACUAAUUAGAGUCUGGCUGAUAUGGUUUGACACAGAGCUAAAUCAUAUGAACCAAACUCUAAUUAGUCAAUAAUUUCUGUU>hsa-mir-5008 MI0017876GGGCUGACCCCUAGGGUCAGGUGAGGCCCUUGGGGCACAGUGGUGCCAUCUCCCCUGUGCUCCCAGGGCCUCGCCUGUCCCUUGAGGUCGGCCC>hsa-mir-5009 MI0017877GACCAGAAGUGUUUUGGAUUUUGGACUUUUUCAGAUUUGGGGAUAUUUGCAUUAUACUUAUCCUAAAUCUGAAAGUCCAAAACCUGAAAUGACCAAUAAG>hsa-mir-5010 MI0017878GAUCCAGGGAACCCUAGAGCAGGGGGAUGGCAGAGCAAAAUUCAUGGCCUACAGCUGCCUCUUGCCAAACUGCACUGGAUUUUGUGUCUCCCAUUCCCCAGAGCUGUCUGAGGUGCUUUG>hsa-mir-5011 MI0017879AGAUGGUAUUGAGUGGAUGCUGUUAUAUAUACAGCCAUGCACUCUGUAGUUUGGGUACACAGUGCAUGGCUGUAUAUAUAACACUAUCCAUUCAUCUUUCAGC>hsa-mir-5047 MI0017932GAAGCGCUUGCCUAGACGAGACACAGUGCAUAAAAACAACUUUUGGGGGACAGGUAUGUUUUCUUGCAGCUGCGGUUGUAAGGUCUUGGCAAGACAAGCA>hsa-mir-5087 MI0017976AGCUUUCUACGGGUUUGUAGCUUUGCUGGCAUGUUAAGUGUUGUCCUACAGUCGCAAGCAUAAGAAAGAGAAAGUA>hsa-mir-5088 MI0017977UCUCCAGGGUGGCCAGGCGGGGCCGGGCCUGAGGGAUGGAGGGGAGCCCAUCAGGGCUCAGGGAUUGGAUGGAGGUGAUGGGGG>hsa-mir-5089 MI0017978AAGGACUUCAGUGGGAUUUCUGAGUAGCAUCCUUGGAAUCUGCACUCAAGGGAUGCUACUCGGAAAUCCCACUGAAGUCCUUUU>hsa-mir-5090 MI0017979UCUGAGGUACCCGGGGCAGAUUGGUGUAGGGUGCAAAGCCUGCCCGCCCCCUAAGCCUUCUGCCCCCAACUCCAGCCUGUCAGGA>hsa-mir-5091 MI0017980GACUGUGGCGACGGAGACGACAAGACUGUGCUGGUCGCGGGUUGUGGGGUUUAGGUCACCGGCAGGGGUCUGGAGUCCCUGGAGGUUAGGGCU>hsa-mir-5092 MI0017981AUCCCAGAUCAGAUGCCAAAGCCAGUGGGGACUGGACAACAUGAUGAGCCCAAACCAAUCCACGCUGAGCUUGGCAUCUGAUUUGGGA>hsa-mir-5093 MI0017982CCCGCCAGGUCCACAUGCCAGAGUGUCAACGUGACCCAGCCAGCCUCCUUCCUGAGCUAGGAGGAUUAGGAAAUGAGGCUGGCUAGGAGCACAGCCAGGG>hsa-mir-5094 MI0017983AAAAGAAAAAAAUCAGUGAAUGCCUUGAACCUAACACACUGCCUUUUAUGUGGUAGGUACAGUGGGCUCACUGAAACAUUCAACU>hsa-mir-5095 MI0018001CUGGGAUUACAGGCGUGAACCACCGCGCCCGGCCUAACUUUUAAGAAACGUCGGCCCGGGAGCGGUGGCUCACGCCUGUAAUCCCAGC>hsa-mir-5096 MI0018004AGUAGAGGUGGGGUUUCACCAUGUUGGUCAGGCUGGUCUCAAACUCCUGACCUCAGGUGAUCCAUCCACC>hsa-mir-5100 MI0019116CCAUGAGGAGCUGGCAGUGGGAUGGCCUGGGGGUAGGAGCGUGGCUUCUGGAGCUAGACCACAUGGGUUCAGAUCCCAGCGGUGCCUCUAACUGGCCACAGGACCUUGGGCAGUCAGCU>hsa-mir-5186 MI0018165UCAGCCAGCUUAUGACUUGACCCUCUCACCUGAUUUCUACCAACCUUUCCUCAGCUGAUUUCUUUCUGGGGAGAGAUUGGUAGAAAUCAGGUGAGAGGGUCAUGCCAUAAGCUGGCUAAC>hsa-mir-5187 MI0018166GACUAAGGGUGGGAUGAGGGAUUGAAGUGGAGCAGGAAUGCGCUUUUCUCCACUGAAUCCUCUUUUCCUCAGGUGG>hsa-mir-5188 MI0018167GGGAGGCAUGGAAAUUUCUCUGGUUUCAAUGGGUACGAUUAUUGUAAGCAGGAUCCAUUCAAUAAUCGGACCCAUUUAAACCGGAGAUUUUAAAAGACAGGAAUAGAAUCCCA>hsa-mir-5189 MI0018168GGCCCGCCUUUUAGGGGCCUCGCUGUCUGGGCACAGGCGGAUGGACAGGCUGGCCUCUGGAUGACCUGCCAACCGUCAGAGCCCAGACCCACGUGGCCUCAGUUGGGGACCAGG>hsa-mir-5190 MI0018169GGUCAUACCCUGGCUCCAGCCCUGUCACAUGGUUAAUGUUCCACAGCCAGUGACUGAGCUGGAGCCAGGGCCACUGCCCC>hsa-mir-5191 MI0018170AGUUGGCCAGGACCCCAAGCCCCCAGCACUUCAUUCUUGCUGUCCUCUCCUGGUCUGGGAGGAUAGAAGAGAGGAUAGGAAGAAUGAAGUGCUGGGCGCUUAGGGGGAUCCUGGCCAACU>hsa-mir-5192 MI0018171UUAGUUCCAGCCUCCUGGCUCACCUGGAACCAUUUCUCCUGGGAAGCAUGGUAGCCAGGAGAGUGGAUUCCAGGUGGUGAGGGCUUGGUACU>hsa-mir-5193 MI0018172CCUAGGAAAGGCUGCUGGUAACUGGGAUGGGGGUUGGGGGGAGGUAAGAAGUCUCUGACUCCUCCUCUACCUCAUCCCAGUUCCAUCACCUGAAGUGGACCUCUUGGGA>hsa-mir-5194 MI0018173AUUUCUUUGGGUUAACUUAAACUCAGCCCUUCUAGGCCCAUUCUUUUCACUCAGGAAUUGGAUAAGCUUUUCUGAGGGGUUUGGAAUGGGAUGGCAGGGAGAGUCACCAGACACCAUGAA>hsa-mir-5195 MI0018174GAGCAAAAACCAGAGAACAACAUGGGAGCGUUCCUAACCCCUAAGGCAACUGGAUGGGAGACCUGACCCAUCCAGUUCUCUGAGGGGGCUCUUGUGUGUUCUACAAGGUUGUUCA>hsa-mir-5196 MI0018175UCUGAGGAGACCUGGGCUGUCAGAGGCCAGGGAAGGGGACGAGGGUUGGGGAACAGGUGGUUAGCACUUCAUCCUCGUCUCCCUCCCAGGUUAGAAGGGCCCCCCUCUCUGAAGG>hsa-mir-5197 MI0018176UAUGGGAUUCCACAGACAAUGAGUAUCAAUGGCACAAACUCAUUCUUGAAUUUUUGCCAGUUCAAGAAGAGACUGAGUCAUCGAAUGCUCUAAAUGUCACUUCACCUCAUGU>hsa-mir-5571 MI0019115AUCUGACACAAAAUGUGAACCAAGCAAUUCUCAAAGGAGCCUCCCAGGAAAUUCACUUUAGGAAGUCCUAGGAGGCUCCUCUGAGAGUUGCUAAAACAAAACAUUGAGAGUCC>hsa-mir-5572 MI0019117AGCCAGACAAGAGGGUCAUGGGGAGUCACUGUCAACCCAGAGCAGGCACUGCCCCUGCGACCAGCCUGGGGCAUCGGUUGGGGUGCAGGGGUCUGCUGGUGAUGCUUUCCAUCUCUUUGCUUUGUCCUGAUUGUAGC>hsa-mir-5579 MI0019133UAUGGUACUCCUUAAGCUAACAGGCCCCUGUCACCAUUAGCUUAAGGAGUACCAGAUC>hsa-mir-5580 MI0019135UGCUGGCUCAUUUCAUAUGUGUGCUGAGAAAAUUCACACAUAUGAAGUGAGCCAGCAC>hsa-mir-5581 MI0019136AGCCUUCCAGGAGAAAUGGAGACCCUAUACAUACCUGUUUCCAUGCCUCCUAGAAGUUCC>hsa-mir-5582 MI0019138UAGGCACACUUAAAGUUAUAGCUACAUCAGUUAUAACUAUAUCAGUUAAAACUUUAAGUGUGCCUAGG>hsa-mir-5583-1 MI0019139AAACUAAUAUACCCAUAUUCUGGCUAGGUGAUCAUCAGAAUAUGGGUAUAUUAGUUUGG>hsa-mir-5583-2 MI0019140AAACUAAUAUACCCAUAUUCUGAUGAUCACCUAGCCAGAAUAUGGGUAUAUUAGUUUGG>hsa-mir-5584 MI0019141CAGGGAAAUGGGAAGAACUAGAUUUGAAUCCAGACCUUUAGUUCUUCCCUUUGCCCAAUU>hsa-mir-5585 MI0019142UGAAGUACCAGCUACUCGAGAGGUCAGAGGAUUGCUCCUGAAUAGCUGGGACUACAGGU>hsa-mir-5586 MI0019143UAUCCAGCUUGUUACUAUAUGCUUUUUAAAUGGGGCACAGAGUGACAAGCUGGUUAAAG>hsa-mir-5587 MI0019144AUGGUCACCUCCGGGACUCAGCCCUGUGCUGAGCCCCGGGCAGUGUGAUCAUC>hsa-mir-5588 MI0019147ACUGGCAUUAGUGGGACUUUUUUUUUUUUUUUUUUUAAUGUUAAAAGUCCCACUAAUGCCAGC>hsa-mir-5589 MI0019148GGCUGGGUGCUCUUGUGCAGUGAGCAACCUACACAACUGCACAUGGCAACCUAGCUCCCA>hsa-mir-5590 MI0019150UUGCCAUACAUAGACUUUAUUGUGUUGAUCAACAAUAAAGUUCAUGUAUGGCAA>hsa-mir-5591 MI0019151UGGGAGCUAAGCUAUGGGUAUACUGAGCUUAUGUAUGCAUCUGCAUACCCAUAGCUUAGCUCCCA>hsa-mir-5680 MI0019280
GCAUUGGGUUAGCAGGUUAGCCCAGCAUUUCCCUUCCUGGACACACAGGAGGAGAAAUGCUGGACUAAUCUGCUAAUCCAAUGC>hsa-mir-5681a MI0019281AGUUUUUGAAGAGUAUUGCCACCCUUUCUAGUCCCUAUUAGACUAGAAAGGGUGGCAAUACCUCUUCCAAAAACU>hsa-mir-5681b MI0019293GAAGAGGUAUUGCCACCCUUUCUAGUCUAAUAGGGACUAGAAAGGGUGGCAAUACUCUUC>hsa-mir-5682 MI0019282GGCCCAUGGGUCUUAUCCUGCAAGGUGCUGCAGAGACGAGGCCUGUAGCACCUUGCAGGAUAAGGUCUACUGGGCC>hsa-mir-5683 MI0019284GGAGCUUGUUACAGAUGCAGAUUCUCUGACUUCUUACUGCACCAGUGAAGUCAGGAUCUGCAUUUGAAUAAGACCC>hsa-mir-5684 MI0019285GCUGAACUCUAGCCUGAGCAACAGAGUGAGAUGGUCUUGUUUUGUUGCCCAGGCUGGAGUCCAGU>hsa-mir-5685 MI0019287CUCUACAUCACAGCCCAGCAGUUAUCACGGGCCCCUCCCCUCAAUGGGCCCGUGAUAACUGCAGGGCUGUGAUGUAGAG>hsa-mir-5686 MI0019290UAUCGUAUCGUAUCGUAUCGUAUUGUAUUGUACUGUAUUGUAUUGUACUGUAUUGUAUCGUAUCGUAUCGUAUCGUAUCGUA>hsa-mir-5687 MI0019291CCUCACUUAUCUGACUCUGAAAUCUUCUAAAUGGUACCCACUUUAUUUAGAACGUUUUAGGGUCAAAUAAGUACAGG>hsa-mir-5688 MI0019292GAAACACUUUGCCUUUUUACAGGAGUUUAUUAUGUUUUGGACAUAGAAACAUAACAAACACCUGUAAAACAGCAAAGUGUUUC>hsa-mir-5689 MI0019294AGCGUGGUAGCAUACACCUGUAGUCCUAGAUACUCAGGAGGGUGAGUAUCUAGGACUACAGGUGUGUGCUACCACGCU>hsa-mir-5690 MI0019295CUUUUAAUUUCAGCUACUACCUCUAUUAGGAUUUGGGAGUUAUACUAAUAGAGGUAAUAGUUGAAAUUAAGAG>hsa-mir-5691 MI0019296GGACAAGCUUGCUCUGAGCUCCGAGAAAGCUGACAGACAGCUGCUUGGUGUUCAGAGCUUGUCUGUCC>hsa-mir-5692a-1 MI0019297GACAGUACAAAUAAUACCACAGUGGGUGUACCUCAUGUGUGUACACCCUGUGAUAUUAUUUGUAAUAUC>hsa-mir-5692a-2 MI0019298UACAAAUAAUACCACAGUGGGUGUACCUCAUGUGUGUACACCCUGUGAUAUUAUUUGUA>hsa-mir-5692b MI0019311GAUAUUAUGAAUAAUAUCACAGUAGGUGUUCACACAUAAUGUGUACACCAUGUGUGUACACCCAUGUGAUAUUUGAAGUAGUAUGUC>hsa-mir-5692c-1 MI0019288UAUAACAUUGUAUAUACCCACUGUGAUAUUAAGAGUAAUAGCUCUCUAGGUUAUUAUGAAUAAUAUCACAGUAGGUGUACACAAUGUUGUA>hsa-mir-5692c-2 MI0019289UGUGUACACCAACUGUGAUAUUAGGAGUCCUAUUUAUUUUUAGGAUAUUAGGAAUAAUAUCACAGUAGGUGUACACA>hsa-mir-5693 MI0019300CUGGGAAGUUAGUUCAUUUCAGUCUGUGCUGUGAGCUAGCCAGCAGUGGCUCUGAAAUGAACUCAAACUCUAG>hsa-mir-5694 MI0019301GCCAACUGCAGAUCAUGGGACUGUCUCAGCCCCAUAUGUAUCUGAAGGCUGAGAAGUCCCAUGAUCCGCACUUGGC>hsa-mir-5695 MI0019302CAAGGCCUAUCUAUCUAGAUUCUUCUUGGCCUCUCUGAGCAUGCAUUCCUGAGACUCCAAGAAGAAUCUAGACAGAUAGGCCUUG>hsa-mir-5696 MI0019303GUGCUCAUUUAAGUAGUCUGAUGCCUACUACUGAUGACAUACAAUGUAAGUGCUCAUUUAGGCGUCAGACUACCUAAAUGAGCAC>hsa-mir-5697 MI0019304AGCAUAUUCUCAAGUAGUUUCAUGAUAAAGGGUGUAUGAGAGAUCAACCCUUUAUCAUGAAACGCUUGAGGAUACGCU>hsa-mir-5698 MI0019305CUGUGCACCUGGGGGAGUGCAGUGAUUGUGGAAUGCAAAGUCCCACAAUCACUGUACUCCCCAGGUGCACAG>hsa-mir-5699 MI0019306CUGUACCCCUGCCCCAACAAGGAAGGACAAGAGGUGUGAGCCACACACACGCCUGGCCUCCUGUCUUUCCUUGUUGGAGCAGGGAUGUAG>hsa-mir-5700 MI0019307UUAAUUAAUGCAUUAAAUUAUUGAAGGCCCUUGGGCACCCCAGGCCUUCAAUAAUUUAAUGCAUUUAUUGA>hsa-mir-5701-1 MI0019308GAUUGGACUUUAUUGUCACGUUCUGAUUGGUUAGCCUAAGACUUGUUCUGAUCCAAUCAGAACAUGAAAAUAACGUCCAAUC>hsa-mir-5701-2 MI0019593GAUUGGACUUUAUUGUCACGUUCUGAUUGGUUAGCCUAAGACUUGUUCUGAUCCAAUCAGAACAUGAAAAUAACGUCCAAUC>hsa-mir-5702 MI0019309GCCUCAACUCCUGGGAUAUGUUGCUGAUCCAACCUGAAAUCCUUCUGUAGGUUGAGUCAGCAACAUAUCCCAUGACUUUUGGGU>hsa-mir-5703 MI0019310UUGCCGUCCCCUUCCUCGUCUUUUCCCCUCAGGAGAAGUCGGGAAGGUGGCGGCGG>hsa-mir-5704 MI0019312UGAUCUUGUUUAGGCCAUCAUCCCAUUAUGCUAAGUCCAUGGGCAAACAUAACAGGAUGAUGGCCUAAACAAGACCA>hsa-mir-5705 MI0019313UCCCCAUUUACACAGGCCAUGAGCCCCGAAACACCCAUCCCAGGAUUGCUGAUGGGUGUUUCGGGGCUCAUGGCCUGUGUAAAUGGGGA>hsa-mir-5706 MI0019314AGCUAGGUCUUCUGGAUAACAUGCUGAAGCUUCUACGUCAUUCAGCACUUGCUUCAGCAUGUUUUCCAGAGGAUCUAGCU>hsa-mir-5707 MI0019315UGUAAGAACACGUUUGAAUGCUGUACAAGGCACAUAUGUGAACAUUGUACCACAUGUACAGCUUUCAAACAUGCUCUUAUA>hsa-mir-5708 MI0019316AUUACAGACAUGAGCGACUGUGCCUGACCAAAAGUCAACAUUAAACAACAAAUCUUGGCCAGGCACAGUGGCUCAUGCCUGUAAU The specification described above is considered to be sufficient for those skilled in the art to enable the practice of the present invention. As the present embodiment is intended as an example of one embodiment of the invention, the invention is not limited to the scope of the provided examples, and other functionally equivalent embodiments are the present invention. It is within the scope of the invention. In addition to those shown and described herein, various modifications of the invention will be apparent to those of skill in the art from the previous specification and will be within the scope of the appended claims. The benefits and objects of the invention are not necessarily included by each embodiment of the invention.

Attachment (A)

Attachment (B)

Attachment (C)

Representative human miRNA sequence

> Hsa-let-7a-1 MI0000060UGGGAUGAGGUAGUAGGUUGUAUAGUUUUAGGGUCACACCCACCACUGGGAGAUAACUAUACAAUCUACUGUCUUUCCUA> hsa-let-7a-2 MI0000061AGGUUGAGGUAGUAGGUUGUAUAGUUUAGAAUUACAUCAAGGGAGAUAACUGUACAGCCUCCUAGCUUUCCU> hsa-let-7a-3 MI0000062GGGUGAGGUAGUAGGUUGUAUAGUUUGGGGCUCUGCCCUGCUAUGGGAUAACUAUACAAUCUACUGUCUUUCCU> hsa-let-7b MI0000063CGGGGUGAGGUAGUAGGUUGUGUGGUUUCAGGGCAGUGAUGUUGCCCCUCGGAAGAUAACUAUACAACCUACUGCCUUCCCUG> hsa-let-7c MI0000064GCAUCCGGGUUGAGGUAGUAGGUUGUAUGGUUUAGAGUUACACCCUGGGAGUUAACUGUACAACCUUCUAGCUUUCCUUGGAGC> hsa-let-7d MI0000065CCUAGGAAGAGGUAGUAGGUUGCAUAGUUUUAGGGCAGGGAUUUUGCCCACAAGGAGGUAACUAUACGACCUGCUGCCUUUCUUAGG> hsa -let-7e MI0000066CCCGGGCUGAGGUAGGAGGUUGUAUAGUUGAGGAGGACACCCAAGGAGAUCACUAUACGGCCUCCUAGCUUUCCCCAGG> hsa-let-7f-1 MI0000067UCAGAGUGAGGUAGUAGAUUGUAUAGUUGUGGGGUAGUGAUUUUACCCUGUUCAGGAGAUAACUAUACAAUCUAUUGCCUUCCCUGA> hsa-let-7f-2 MI0000068UGUGGGAUGAGGUAGUAGAUUGUAUAGUUUUAGGGUCAUACCCCAUCUUGGAGAUAACUAUACAGUCUACUGUCUUUCCCACG> hsa-let-7g MI0000433AGGCUGAGGUAGUAGUUUGUACAGUUUGAGGGUCUAUGAUACCACCCGGUA CAGGAGAUAACUGUACAGGCCACUGCCUUGCCA> hsa-let-7i MI0000434CUGGCUGAGGUAGUAGUUUGUGCUGUUGGUCGGGUUGUGACAUUGCCCGCUGUGGAGAUAACUGCGCAAGCUACUGCCUUGCUA> hsa-mir-1-1 MI0000651UGGGAAACAUACUUCUUUAUAUGCCCAUAUGGACCUGCUAAGCUAUGGAAUGUAAAGAAGUAUGUAUCUCA> hsa-mir-1-2 MI0000437ACCUACUCAGAGUACAUACUUCUUUAUGUACCCAUAUGAACAUACAAUGCUAUGGAAUGUAAAGAAGUAUGUAUUUUUGGUAGGC> hsa-mir-7-1 MI0000263UUGGAUGUUGGCCUAGUUCUGUGUGGAAGACUAGUGAUUUUGUUGUUUUUAGAUAACUAAAUCGACAACAAAUCACAGUCUGCCAUAUGGCACAGGCCAUGCCUCUACAG> hsa-mir-7-2 MI0000264CUGGAUACAGAGUGGACCGGCUGGCCCCAUCUGGAAGACUAGUGAUUUUGUUGUUGUCUUACUGCGCUCAACAACAAAUCCCAGUCUACCUAAUGGUGCCAGCCAUCGCA> hsa-mir-7 -3 MI0000265AGAUUAGAGUGGCUGUGGUCUAGUGCUGUGUGGAAGACUAGUGAUUUUGUUGUUCUGAUGUACUACGACAACAAGUCACAGCCGGCCUCAUAGCGCAGACUCCCUUCGAC> hsa-mir-9-1 MI0000466CGGGGUUGGUUGUUAUCUUUGGUUAUCUAGCUGUAUGAGUGGUGUGGAGUCUUCAUAAAGCUAGAUAACCGAAAGUAAAAAUAACCCCA> hsa-mir-9-2 MI0000467GGAAGCGAGUUGUUAUCUUUGGUUAUCUAGCUGUAUGAGUGUAUUGGUCUUCAUAAAGCUAGAUAACCGAAAGUAAAAACUCCUUCA> hsa-mir-9-3 MI0000468GGAGGCCCGUUUCUCUCUUUGGUU AUCUAGCUGUAUGAGUGCCACAGAGCCGUCAUAAAGCUAGAUAACCGAAAGUAGAAAUGAUUCUCA> hsa-mir-10a MI0000266GAUCUGUCUGUCUUCUGUAUAUACCCUGUAGAUCCGAAUUUGUGUAAGGAAUUUUGUGGUCACAAAUUCGUAUCUAGGGGAAUAUGUAGUUGACAUAAACACUCCGCUCU> hsa-mir-10b MI0000267CCAGAGGUUGUAACGUUGUCUAUAUAUACCCUGUAGAACCGAAUUUGUGUGGUAUCCGUAUAGUCACAGAUUCGAUUCUAGGGGAAUAUAUGGUCGAUGCAAAAACUUCA> hsa-mir-15a MI0000069CCUUGGAGUAAAGUAGCAGCACAUAAUGGUUUGUGGAUUUUGAAAAGGUGCAGGCCAUAUUGUGCUGCCUCAAAAAUACAAGG> hsa-mir-15b MI0000438UUGAGGCCUUAAAGUACUGUAGCAGCACAUCAUGGUUUACAUGCUACAGUCAAGAUGCGAAUCAUUAUUUGCUGCUCUAGAAAUUUAAGGAAAUUCAU> hsa-mir-16-1 MI0000070GUCAGCAGUGCCUUAGCAGCACGUAAAUAUUGGCGUUAAGAUUCUAAAAUUAUCUCCAGUAUUAACUGUGCUGCUGAAGUAAGGUUGAC> hsa-mir-16-2 MI0000115GUUCCACUCUAGCAGCACGUAAAUAUUGGCGUAGUGAAAUAUAUAUUAAACACCAAUAUUACUGUGCUGCUUUAGUGUGAC> hsa- mir-17 MI0000071GUCAGAAUAAUGUCAAAGUGCUUACAGUGCAGGUAGUGAUAUGUGCAUCUACUGCAGUGAAGGCACUUGUAGCAUUAUGGUGAC> hsa-mir-18a MI0000072UGUUCUAAGGUGCAUCUAGUCGAUAGUGAAGUAGAUUAGCAUCUACUGCCCUAG GUGCAUCUAGUGCAGUUAGUGAAGCAGCUUAGAAUCUACUGCCCUAAAUGCCCCUUCUGGCA> hsa-mir-19a MI0000073GCAGUCCUCUGUUAGUUUUGCAUAGUUGCACUACAAGAAGAAUGUAGUUGUGCAAAUCUAUGCAAAACUGAUGGUGGCCUGC> hsa-mir-19b-1 MI0000074CACUGUUCUAUGGUUAGUUUUGCAGGUUUGCAUCCAGCUGUGUGAUAUUCUGCUGUGCAAAUCCAUGCAAAACUGACUGUGGUAGUG> hsa-mir-19b-2 MI0000075ACAUUGCUACUUACAAUUAGUUUUGCAGGUUUGCAUUUCAGCGUAUAUAUGUAUAUGUGGCUGUGCAAAUCCAUGCAAAACUGAUUGUGAUAAUGU> hsa-mir-20a MI0000076GUAGCACUAAAGUGCUUAUAGUGCAGGUAGUGUUUAGUUAUCUACUGCAUUAUGAGCACUUAAAGUACUGC> hsa-mir-20b MI0001519AGUACCAAAGUGCUCAUAGUGCAGGUAGUUUUGGCAUGACUCUACUGUAGUAUGGGCACUUCCAGUACU> hsa-mir-21 MI0000077UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGCAACACCAGUCGAUGGGCUGUCUGACA> hsa- mir-22 MI0000078GGCUGAGCCGCAGUAGUUCUUCAGUGGCAAGCUUUAUGUCCUGACCCAGCUAAAGCUGCCAGUUGAAGAACUGUUGCCCUCUGCC> hsa-mir-23a MI0000079GGCCGGCUGGGGUUCCUGGGGAUGGGAUUUGCUUCCUGUCACAAAUCACAUUGCCAGGGAUUUCCAACCGACC> hsa-mir-23b MI0000439CUCAGGUGCUCUGGCUGCUUGGGUUCCUGGCAUGCUGAUUUGUGACUUAAGAUUAAAAUCACAUUGCCAGGGAUUACCACGCAACCACGACCUUGGC> hsa-m ir-23c MI0016010AGUGACUUUCCAGGUGUCACACAGUGAGUGGCAUAAUCAGAGUACAAUUUGAGUCAUGCCCAUACAUCACAUUGCCAGUGAUUACCCAAGGAAAGUGACG> hsa-mir-24-1 MI0000080CUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCAUUUUACACACUGGCUCAGUUCAGCAGGAACAGGAG> hsa-mir-24-2 MI0000081CUCUGCCUCCCGUGCCUACUGAGCUGAAACACAGUUGGUUUGUGUACACUGGCUCAGUUCAGCAGGAACAGGG> hsa-mir-25 MI0000082GGCCAGUGUUGAGAGGCGGAGACUUGGGCAAUUGCUGGACGCUGCCCUGGGCAUUGCACUUGUCUCGGUCUGACAGUGCCGGCC> hsa-mir-26a-1 MI0000083GUGGCCUCGUUCAAGUAAUCCAGGAUAGGCUGUGCAGGUCCCAAUGGGCCUAUUCUUGGUUACUUGCACGGGGACGC> hsa-mir-26a-2 MI0000750GGCUGUGGCUGGAUUCAAGUAAUCCAGGAUAGGCUGUUUCCAUCUGUGAGGCCUAUUCUUGAUUACUUGUUUCUGGAGGCAGCU> hsa- mir-26b MI0000084CCGGGACCCAGUUCAAGUAAUUCAGGAUAGGUUGUGUGCUGUCCAGCCUGUUCUCCAUUACUUGGCUCGGGGACCGG> hsa-mir-27a MI0000085CUGAGGAGCAGGGCUUAGCUGCUUGUGAGCAGGGUCCACACCAAGUCGUGUUCACAGUGGCUAAGUUCCGCCCCCCAG> hsa-mir-27b MI0000440ACCUCUCUAACAAGGUGCAGAGCUUAGCUGAUUGGUGAACAGUGAUUGGUUUCCGCUUUGUUCACAGUGGCUAAGUUCUGCACCUGAAGAGAAGGUG> hsa-mir-28 MI0000086GGUCCUUGCCCUCAAGGAGCUCACAGUCUAUUGAGUUACCUUUC UGACUUUCCCACUAGAUUGUGAGCUCCUGGAGGGCAGGCACU> hsa-mir-29a MI0000087AUGACUGAUUUCUUUUGGUGUUCAGAGUCAAUAUAAUUUUCUAGCACCAUCUGAAAUCGGUUAU> hsa-mir-29b-1 MI0000105CUUCAGGAAGCUGGUUUCAUAUGGUGGUUUAGAUUUAAAUAGUGAUUGUCUAGCACCAUUUGAAAUCAGUGUUCUUGGGGG> hsa-mir-29b-2 MI0000107CUUCUGGAAGCUGGUUUCACAUGGUGGCUUAGAUUUUUCCAUCUUUGUAUCUAGCACCAUUUGAAAUCAGUGUUUUAGGAG> hsa-mir-29c MI0000735AUCUCUUACACAGGCUGACCGAUUUCUCCUGGUGUUCAGAGUCUGUUUUUGUCUAGCACCAUUUGAAAUCGGUUAUGAUGUAGGGGGA> hsa-mir-30a MI0000088GCGACUGUAAACAUCCUCGACUGGAAGCUGUGAAGCCACAGAUGGGCUUUCAGUCGGAUGUUUGCAGCUGC> hsa-mir-30b MI0000441ACCAAGUUUCAGUUCAUGUAAACAUCCUACACUCAGCUGUAAUACAUGGAUUGGCUGGGAGGUGGAUGUUUACUUCAGCUGACUUGGA> hsa- mir-30c-1 MI0000736ACCAUGCUGUAGUGUGUGUAAACAUCCUACACUCUCAGCUGUGAGCUCAAGGUGGCUGGGAGAGGGUUGUUUACUCCUUCUGCCAUGGA> hsa-mir-30c-2 MI0000254AGAUACUGUAAACAUCCUACACUCUCAGCUGUGGAAAGUAAGAAAGCUGGGAGAAGGCUGUUUACUCUUUCU> hsa-mir-30d MI0000255GUUGUUGUAAACAUCCCCGACUGGAAGCUGUAAGACACAGCUAAGCUUUCAGUCAGAUGUUUGCUGCUAC> hsa-mir-30e MI0000749GGGCAGUCUUUGCUACUGUAAACAUC CUUGACUGGAAGCUGUAAGGUGUUCAGAGGAGCUUUCAGUCGGAUGUUUACAGCGGCAGGCUGCCA> hsa-mir-31 MI0000089GGAGAGGAGGCAAGAUGCUGGCAUAGCUGUUGAACUGGGAACCUGCUAUGCCAACAUAUUGCCAUCUUUCC> hsa-mir-32 MI0000090GGAGAUAUUGCACAUUACUAAGUUGCAUGUUGUCACGGCCUCAAUGCAAUUUAGUGUGUGUGAUAUUUUC> hsa-mir-33a MI0000091CUGUGGUGCAUUGUAGUUGCAUUGCAUGUUCUGGUGGUACCCAUGCAAUGUUUCCACAGUGCAUCACAG> hsa-mir-33b MI0003646GCGGGCGGCCCCGCGGUGCAUUGCUGUUGCAUUGCACGUGUGUGAGGCGGGUGCAGUGCCUCGGCAGUGCAGCCCGGAGCCGGCCCCUGGCACCAC> hsa-mir-34a MI0000268GGCCAGCUGUGAGUGUUUCUUUGGCAGUGUCUUAGCUGGUUGUUGUGAGCAAUAGUAAGGAAGCAAUCAGCAAGUAUACUGCCCUAGAAGUGCUGCACGUUGUGGGGCCC> hsa-mir-34b MI0000742GUGCUCGGUUUGUAGGCAGUGUCAUUAGCUGAUUGUACUGUGGUGGUUACAAUCACUAACUCCACUGCCAUCAAAACAAGGCAC> hsa-mir-34c MI0000743AGUCUAGUUACUAGGCAGUGUAGUUAGCUGAUUGCUAAUAGUACCAAUCACUAACCACACGGCCAGGUAAAAAGAUU > hsa-mir-92a-1 MI0000093CUUUCUACAGGUUGGGAUCGGUUGCAAUGCUGUGUUUCUGUAUGGUAUUGCACUUGUCCCGGCCUGUUGAGUUUGG> hsa-mir-92a-2 MI0000094UCAUCCCUGGGUGGGGAUUUGUUGCAUUCUGUGUUCUC -mir-92b MI0003560CGGGCCCCGGGCGGGCGGGAGGGACGGGACGCGGUGCAGUGUUGUUUUUUCCCCCGCCAAUAUUGCACUCGUCCCGGCCUCCGGCCCCCCCGGCCC> hsa-mir-93 MI0000095CUGGGGGCUCCAAAGUGCUGUUCGUGCAGGUAGUGUGAUUACCCAACCUACUGCUGAGCUAGCACUUCCCGAGCCCCCGG> hsa-mir-95 MI0000097AACACAGUGGGCACUCAAUAAAUGUCUGUUGAAUUGAAAUGCGUUACAUUCAACGGGUAUUUAUUGAGCACCCACUCUGUG> hsa-mir-96 MI0000098UGGCCGAUUUUGGCACUAGCACAUUUUUGCUUGUGUCUCUCCGCUCUGAGCAAUCAUGUGCAGUGCCAAUAUGGGAAA> hsa-mir-98 MI0000100AGGAUUCUGCUCAUGCCAGGGUGAGGUAGUAAGUUGUAUUGUUGUGGGGUAGGGAUAUUAGGCCCCAAUUAGAAGAUAACUAUACAACUUACUACUUUCCCUGGUGUGUGGCAUAUUCA> hsa-mir-99a MI0000101CCCAUUGGCAUAAACCCGUAGAUCCGAUCUUGUGGUGAAGUGGACCGCACAAGCUCGCUUCUAUGGGUCUGUGUCAGUGUG> hsa-mir-99b MI0000746GGCACCCACCCGUAGAACCGACCUUGCGGGGCCUUCGCCGCACACAAGCUCGUGUCUGUGGGUCCGUGUC> hsa- mir-100 MI0000102CCUGUUGCCACAAACCCGUAGAUCCGAACUUGUGGUAUUAGUCCGCACAAGCUUGUAUCUAUAGGUAUGUGUCUGUUAGG> hsa-mir-101-1 MI0000103UGCCCUGGCUCAGUUAUCACAGUGCUGAUGCUGUCUAUUCUAAAGGUACAGUACUGUGAU UACCGAUGCUGUAUAUCUGAAAGGUACAGUACUGUGAUAACUGAAGAAUGGUGGU> hsa-mir-103a-1 MI0000109UACUGCCCUCGGCUUCUUUACAGUGCUGCCUUGUUGCAUAUGGAUCAAGCAGCAUUGUACAGGGCUAUGAAGGCAUUG> hsa-mir-103a-2 MI0000108UUGUGCUUUCAGCUUCUUUACAGUGCUGCCUUGUAGCAUUCAGGUCAAGCAGCAUUGUACAGGGCUAUGAAAGAACCA> hsa-mir-103b-1 MI0007261UCAUAGCCCUGUACAAUGCUGCUUGAUCCAUAUGCAACAAGGCAGCACUGUAAAGAAGCCGA> hsa-mir-103b-2 MI0007262UCAUAGCCCUGUACAAUGCUGCUUGACCUGAAUGCUACAAGGCAGCACUGUAAAGAAGCUGA> hsa-mir-105-1 MI0000111UGUGCAUCGUGGUCAAAUGCUCAGACUCCUGUGGUGGCUGCUCAUGCACCACGGAUGUUUGAGCAUGUGCUACGGUGUCUA> hsa-mir -105-2 MI0000112UGUGCAUCGUGGUCAAAUGCUCAGACUCCUGUGGUGGCUGCUUAUGCACCACGGAUGUUUGAGCAUGUGCUAUGGUGUCUA> hsa-mir-106a MI0000113CCUUGGCCAUGUAAAAGUGCUUACAGUGCAGGUAGCUUUUUGAGAUCUACUGCAAUGUAAGCACUUCUUACAUUACCAUGG> hsa-mir-106b MI0000734CCUGCCGGGGCUAAAGUGCUGACAGUGCAGAUAGUGGUCCUCUCCGUGCUACCGCACUGUGGGUACUUGCUGCUCCAGCAGG> hsa-mir-107 MI0000114CUCUCUGCUUUCAGCUUCUUUACAGUGUUGCCUUGUGGCAUGGAGUUCAAGCAGCAUUGUACAGGGCUAUCAAAGCACAGA> hsa-mir-122 MI0000442CCUUAGCAGAGCUGUGGAGUG UGACAAUGGUGUUUGUGUCUAAACUAUCAAACGCCAUUAUCACACUAAAUAGCUACUGCUAGGC> hsa-mir-124-1 MI0000443AGGCCUCUCUCUCCGUGUUCACAGCGGACCUUGAUUUAAAUGUCCAUACAAUUAAGGCACGCGGUGAAUGCCAAGAAUGGGGCUG> hsa-mir-124-2 MI0000444AUCAAGAUUAGAGGCUCUGCUCUCCGUGUUCACAGCGGACCUUGAUUUAAUGUCAUACAAUUAAGGCACGCGGUGAAUGCCAAGAGCGGAGCCUACGGCUGCACUUGAA> hsa-mir-124-3 MI0000445UGAGGGCCCCUCUGCGUGUUCACAGCGGACCUUGAUUUAAUGUCUAUACAAUUAAGGCACGCGGUGAAUGCCAAGAGAGGCGCCUCC> hsa-mir-125a MI0000469UGCCAGUCUCUAGGUCCCUGAGACCCUUUAACCUGUGAGGACAUCCAGGGUCACAGGUGAGGUUCUUGGGAGCCUGGCGUCUGGCC> hsa-mir-125b-1 MI0000446UGCGCUCCUCUCAGUCCCUGAGACCCUAACUUGUGAUGUUUACCGUUUAAAUCCACGGGUUAGGCUCUUGGGAGCUGCGAGUCGUGCU> hsa-mir-125b -2 MI0000470ACCAGACUUUUCCUAGUCCCUGAGACCCUAACUUGUGAGGUAUUUUAGUAACAUCACAAGUCAGGCUCUUGGGACCUAGGCGGAGGGGA> hsa-mir-126 MI0000471CGCUGGCGACGGGACAUUAUUACUUUUGGUACGCGCUGUGACACUUCAAACUCGUACCGUGAGUAAUAAUGCGCCGUCCACGGCA> hsa-mir-127 MI0000472UGUGAUCACUGUCUCCAGCCUGCUGAAGCUCAGAGGGCUCUGAUUCAGAAAGAUCAUCGGAUCCGUCUGAGCUUGGC
UGGUCGGAAGUCUCAUCAUC> hsa-mir-128-1 MI0000447UGAGCUGUUGGAUUCGGGGCCGUAGCACUGUCUGAGAGGUUUACAUUUCUCACAGUGAACCGGUCUCUUUUUCAGCUGCUUC> hsa-mir-128-2 MI0000727UGUGCAGUGGGAAGGGGGGCCGAUACACUGUACGAGAGUGAGUAGCAGGUCUCACAGUGAACCGGUCUCUUUCCCUACUGUGUC> hsa-mir-129-1 MI0000252GGAUCUUUUUGCGGUCUGGGCUUGCUGUUCCUCUCAACAGUAGUCAGGAAGCCCUUACCCCAAAAAGUAUCU> hsa-mir-129-2 MI0000473UGCCCUUCGCGAAUCUUUUUGCGGUCUGGGCUUGCUGUACAUAACUCAAUAGCCGGAAGCCCUUACCCCAAAAAGCAUUUGCGGAGGGCG> hsa-mir-130a MI0000448UGCUGCUGGCCAGAGCUCUUUUCACAUUGUGCUACUGUCUGCACCUGUCACUAGCAGUGCAAUGUUAAAAGGGCAUUGGCCGUGUAGUG> hsa-mir-130b MI0000748GGCCUGCCCGACACUCUUUCCCUGUUGCACUACUAUAGGCCGCUGGGAAGCAGUGCAAUGAUGAAAGGGCAUCGGUCAGGUC> hsa-mir-132 MI0000449CCGCCCCCGCGUCUCCAGGGCAACCGUGGCUUUCGAUUGUUACUGUGGGAACUGGAGGUAACAGUCUACAGCCAUGGUCGCCCCGCAGCACGCCCACGCGC> hsa-mir-133a-1 MI0000450ACAAUGCUUUGCUAGAGCUGGUAAAAUGGAACCAAAUCGCCUCUUCAAUGGAUUUGGUCCCCUUCAACCAGCUGUAGCUAUGCAUUGA> hsa-mir-133a-2 MI0000451GGGAGCCAAAUGCUUUGCUAGAGCUGGUAAAAUGGAACCAAAUCGACUGUCCAAUGGAUUUGGUCCCCUUCAACCAGC UGUAGCUGUGCAUUGAUGGCGCCG> hsa-mir-133b MI0000822CCUCAGAAGAAAGAUGCCCCCUGCUCUGGCUGGUCAAACGGAACCAAGUCCGUCUUCCUGAGAGGUUUGGUCCCCUUCAACCAGCUACAGCAGGGCUGGCAAUGCCCAGUCCUUGGAGA> hsa-mir-134 MI0000474CAGGGUGUGUGACUGGUUGACCAGAGGGGCAUGCACUGUGUUCACCCUGUGGGCCACCUAGUCACCAACCCUC> hsa-mir-135a-1 MI0000452AGGCCUCGCUGUUCUCUAUGGCUUUUUAUUCCUAUGUGAUUCUACUGCUCACUCAUAUAGGGAUUGGAGCCGUGGCGCACGGCGGGGACA> hsa-mir-135a-2 MI0000453AGAUAAAUUCACUCUAGUGCUUUAUGGCUUUUUAUUCCUAUGUGAUAGUAAUAAAGUCUCAUGUAGGGAUGGAAGCCAUGAAAUACAUUGUGAAAAAUCA> hsa-mir-135b MI0000810CACUCUGCUGUGGCCUAUGGCUUUUCAUUCCUAUGUGAUUGCUGUCCCAAACUCAUGUAGGGCUAAAAGCCAUGGGCUACAGUGAGGGGCGAGCUCC> hsa-mir-136 MI0000475UGAGCCCUCGGAGGACUCCAUUUGUUUUGAUGAUGGAUUCUUAUGCUCCAUCAUCGUCUCAAAUGAGUCUUCAGAGGGUUCU> hsa- mir-137 MI0000454GGUCCUCUGACUCUCUUCGGUGACGGGUAUCUUGGGUGGAUAAUACGGAUUACGUUGUUAUUGCUUAAGAAUACGCGUAGUCGAGGAGAGUACCAGCGGCA> hsa-mir-138-1 MI0000476CCCUGGCAUGGUGUGGUGGGGCAGCUGGU GCUGCAGCUGGUGUUGUGAAUCAGGCCGACGAGCAGCGCAUCCUCUUACCCGGCUAUUUCACGACACCAGGGUUGCAUCA> hsa-mir-139 MI0000261GUGUAUUCUACAGUGCACGUGUCUCCAGUGUGGCUCGGAGGCUGGAGACGCGGCCCUGUUGGAGUAAC> hsa-mir-140 MI0000456UGUGUCUCUCUCUGUGUCCUGCCAGUGGUUUUACCCUAUGGUAGGUUACGUCAUGCUGUUCUACCACAGGGUAGAACCACGGACAGGAUACCGGGGCACC> hsa-mir-141 MI0000457CGGCCGGCCCUGGGUCCAUCUUCCAGUACAGUGUUGGAUGGUCUAAUUGUGAAGCUCCUAACACUGUCUGGUAAAGAUGGCUCCCGGGUGGGUUC> hsa-mir-142 MI0000458GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAGGGUGUAGUGUUUCCUACUUUAUGGAUGAGUGUACUGUG> hsa-mir-143 MI0000459GCGCAGCGCCCUGUCUCCCAGCCUGAGGUGCAGUGCUGCAUCUCUGGUCAGUUGGGAGUCUGAGAUGAAGCACUGUAGCUCAGGAAGAGAGAAGUUGUUCUGCAGC> hsa-mir-144 MI0000460UGGGGCCCUGGCUGGGAUAUCAUCAUAUACUGUAAGUUUGCGAUGAGACACUACAGUAUAGAUGAUGUACUAGUCCGGGCACCCCC> hsa-mir-145 MI0000461CACCUUGUCCUCACGGUCCAGUUUUCCCAGGAAUCCCUUAGAUGCUAAGAUGGGGAUUCCUGGAAAUACUGUUCUUGAGGUCAUGGUU > hsa-mir-146a MI0000477CCGAUGUGUAUCCUCAGCUUUGAGAACUGAAUUCCAUGGGUUGUGUCAGUGUCAGACCUCUGAAAUUCAGUUCUUCAGCUGGGAUAUCUCUGUCAUCGU> hsa-mir-146b MI0003129CCUGGCACUGAGAACUGAAUUCCAUAGGCUGUGAGCUCUAGCAAUGCCCUGUGGACUCAGUUCUGGUGCCCGG> hsa-mir-147a MI0000262AAUCUAAAGACAACAUUUCUGCACACACACCAGACUAUGGAAGCCAGUGUGUGGAAAUGCUUCUGCUAGAUU> hsa-mir-147b MI0005544UAUAAAUCUAGUGGAAACAUUUCUGCACAAACUAGAUUCUGGACACCAGUGUGCGGAAAUGCUUCUGCUACAUUUUUAGG> hsa-mir-148a MI0000253GAGGCAAAGUUCUGAGACACUCCGACUCUGAGUAUGAUAGAAGUCAGUGCACUACAGAACUUUGUCUC> hsa-mir-148b MI0000811CAAGCACGAUUAGCAUUUGAGGUGAAGUUCUGUUAUACACUCAGGCUGUGGCUCUCUGAAAGUCAGUGCAUCACAGAACUUUGUCUCGAAAGCUUUCUA> hsa-mir-149 MI0000478GCCGGCGCCCGAGCUCUGGCUCCGUGUCUUCACUCCCGUGCUUGUCCGAGGAGGGAGGGAGGGACGGGGGCUGUGCUGGGGCAGCUGGA> hsa-mir-150 MI0000479CUCCCCAUGGCCCUGUCUCCCAACCCUUGUACCAGUGCUGGGCUCAGACCCUGGUACAGGCCUGGGGGACAGGGACCUGGGGAC> hsa-mir-151a MI0000809UUUCCUGCCCUCGAGGAGCUCACAGUCUAGUAUGUCUCAUCCCCUACUAGACUGAAGCUCCUUGAGGACAGGGAUGGUCAUACUCACCUC > hsa-mir-151b MI0003772ACCUCUGAUGUGUCAGUCUCUCUUCAGGGCUCCCGAGACACAGAAACAGACACCUGCCCUCGAGGAGCUCACAGUCUAGACAAACAAACCCAGGGU> hsa-mir-152 MI0000462UGUCCCCCCCGGCCAGGUUCUGUGAUACACUCCGA CUCGGGCUCUGGAGCAGUCAGUGCAUGACAGAACUUGGGCCCGGAAGGACC> hsa-mir-153-1 MI0000463CUCACAGCUGCCAGUGUCAUUUUUGUGAUCUGCAGCUAGUAUUCUCACUCCAGUUGCAUAGUCACAAAAGUGAUCAUUGGCAGGUGUGGC> hsa-mir-153-2 MI0000464AGCGGUGGCCAGUGUCAUUUUUGUGAUGUUGCAGCUAGUAAUAUGAGCCCAGUUGCAUAGUCACAAAAGUGAUCAUUGGAAACUGUG> hsa-mir-154 MI0000480GUGGUACUUGAAGAUAGGUUAUCCGUGUUGCCUUCGCUUUAUUUGUGACGAAUCAUACACGGUUGACCUAUUUUUCAGUACCAA> hsa-mir-155 MI0000681CUGUUAAUGCUAAUCGUGAUAGGGGUUUUUGCCUCCAACUGACUCCUACAUAUUAGCAUUAACAG> hsa-mir-181a-1 MI0000289UGAGUUUUGAGGUUGCUUCAGUGAACAUUCAACGCUGUCGGUGAGUUUGGAAUUAAAAUCAAAACCAUCGACCGUUGAUUGUACCCUAUGGCUAACCAUCAUCUACUCCA> hsa-mir-181a-2 MI0000269AGAAGGGCUAUCAGGCCAGCCUUCAGAGGACUCCAAGGAACAUUCAACGCUGUCGGUGAGUUUGGGAUUUGAAAAAACCACUGACCGUUGACUGUACCUUGGGGUCCUUA> hsa-mir-181b-1 MI0000270CCUGUGCAGAGAUUAUUUUUUAAAAGGUCACAAUCAACAUUCAUUGCUGUCGGUGGGUUGAACUGUGUGGACAAGCUCACUGAACAAUGAAUGCAACUGUGGCCCCGCUU> hsa-mir-181b-2 MI0000683CUGAUGGCUGCACUCAACAUUCAUUGCUGUCGGUGGGUUUGAGUCUGAAUCAACUCACUGAUCAAUGAAUGCAAACUGCGGACCAAACA> hsa-mir-181 c MI0000271CGGAAAAUUUGCCAAGGGUUUGGGGGAACAUUCAACCUGUCGGUGAGUUUGGGCAGCUCAGGCAAACCAUCGACCGUUGAGUGGACCCUGAGGCCUGGAAUUGCCAUCCU> hsa-mir-181d MI0003139GUCCCCUCCCCUAGGCCACAGCCGAGGUCACAAUCAACAUUCAUUGUUGUCGGUGGGUUGUGAGGACUGAGGCCAGACCCACCGGGGGAUGAAUGUCACUGUGGCUGGGCCAGACACGGCUUAAGGGGAAUGGGGAC> hsa-mir-182 MI0000272GAGCUGCUUGCCUCCCCCCGUUUUUGGCAAUGGUAGAACUCACACUGGUGAGGUAACAGGAUCCGGUGGUUCUAGACUUGCCAACUAUGGGGCGAGGACUCAGCCGGCAC> hsa-mir-183 MI0000273CCGCAGAGUGUGACUCCUGUUCUGUGUAUGGCACUGGUAGAAUUCACUGUGAACAGUCUCAGUCAGUGAAUUACCGAAGGGCCAUAAACAGAGCAGAGACAGAUCCACGA> hsa-mir-184 MI0000481CCAGUCACGUCCCCUUAUCACUUUUCCAGCCCAGCUUUGUGACUGUAAGUGUUGGACGGAGAACUGAUAAGGGUAGGUGAUUGA> hsa-mir-185 MI0000482AGGGGGCGAGGGAUUGGAGAGAAAGGCAGUUCCUGAUGGUCCCCUCCCCAGGGGCUGGCUUUCCUCUGGUCCUUCCCUCCCA> hsa-mir-186 MI0000483UGCUUGUAACUUUCCAAAGAAUUCUCCUUUUGGGCUUUCUGGUUUUAUUUUAAGCCCAAAGGUGAAUUUUUUGGGAAGUUUGAGCU> hsa-mir-187 MI0000274GGUCGGGCUCACCAUGACAGUGUGAGACCUCGGGCUACAACACAGGACCCGGGCGCUGCUCUGACCCCUCGUGUCUUGUGUUGCAGCCGGAGGGACGCAGGUCCGCA> hsa-m ir-188 MI0000484UGCUCCCUCUCUCACAUCCCUUGCAUGGUGGAGGGUGAGCUUUCUGAAAACCCCUCCCACAUGCAGGGUUUGCAGGAUGGCGAGCC> hsa-mir-190a MI0000486UGCAGGCCUCUGUGUGAUAUGUUUGAUAUAUUAGGUUGUUAUUUAAUCCAACUAUAUAUCAAACAUAUUCCUACAGUGUCUUGCC> hsa-mir-190b MI0005545UGCUUCUGUGUGAUAUGUUUGAUAUUGGGUUGUUUAAUUAGGAACCAACUAAAUGUCAAACAUAUUCUUACAGCAGCAG> hsa-mir-191 MI0000465CGGCUGGACAGCGGGCAACGGAAUCCCAAAAGCAGCUGUUGUCUCCAGAGCAUUCCAGCUGCGCUUGGAUUUCGUCCCCUGCUCUCCUGCCU> hsa-mir-192 MI0000234GCCGAGACCGAGUGCACAGGGCUCUGACCUAUGAAUUGACAGCCAGUGCUCUCGUCUCCCCUCUGGCUGCCAAUUCCAUAGGUCACAGGUAUGUUCGCCUCAAUGCCAGC> hsa-mir-193a MI0000487CGAGGAUGGGAGCUGAGGGCUGGGUCUUUGCGGGCGAGAUGAGGGUGUCGGAUCAACUGGCCUACAAAGUCCCAGUUCUCGGCCCCCG> hsa-mir-193b MI0003137GUGGUCUCAGAAUCGGGGUUUUGAGGGCGAGAUGAGUUUAUGUUUUAUCCAACUGGCCCUCAAAGUCCCGCUUUUGGGGUCAU> hsa-mir -194-1 MI0000488AUGGUGUUAUCAAGUGUAACAGCAACUCCAUGUGGACUGUGUACCAAUUUCCAGUGGAGAUGCUGUUACUUUGAUGGUUACCAA> hsa-mir-194-2 MI0000732UGGUUCCCGCCCCCUGUAACAGCAACUCCAUGUGGAAGUGCCACUGGUUC r-195 MI0000489AGCUUCCCUGGCUCUAGCAGCACAGAAAUAUUGGCACAGGGAAGCGAGUCUGCCAAUAUUGGCUGUGCUGCUCCAGGCAGGGUGGUG> hsa-mir-196a-1 MI0000238GUGAAUUAGGUAGUUUCAUGUUGUUGGGCCUGGGUUUCUGAACACAACAACAUUAAACCACCCGAUUCAC> hsa-mir-196a-2 MI0000279UGCUCGCUCAGCUGAUCUGUGGCUUAGGUAGUUUCAUGUUGUUGGGAUUGAGUUUUGAACUCGGCAACAAGAAACUGCCUGAGUUACAUCAGUCGGUUUUCGUCGAGGGC> hsa-mir-196b MI0001150ACUGGUCGGUGAUUUAGGUAGUUUCCUGUUGUUGGGAUCCACCUUUCUCUCGACAGCACGACACUGCCUUCAUUACUUCAGUUG> hsa-mir-197 MI0000239GGCUGUGCCGGGUAGAGAGGGCAGUGGGAGGUAAGAGCUCUUCACCCUUCACCACCUUCUCCACCCAGCAUGGCC> hsa-mir-198 MI0000240UCAUUGGUCCAGAGGGGAGAUAGGUUCCUGUGAUUUUUCCUUCUUCUCUAUAGAAUAAAUGA> hsa-mir-199a- 1 MI0000242GCCAACCCAGUGUUCAGACUACCUGUUCAGGAGGCUCUCAAUGUGUACAGUAGUCUGCACAUUGGUUAGGC> hsa-mir-199a-2 MI0000281AGGAAGCUUCUGGAGAUCCUGCUCCGUCGCCCCAGUGUUCAGACUACCUGUUCAGGACAAUGCCGUUGUACAGUAGUCUGCACAUUGGUUAGACUGGGCAAGGGAGAGCA> hsa-mir-199b MI0000282CCAGAGGACACCUCCACUCCGUCUACCCAGUGUUUAGACUAUCUGUUCAGGACUCCCAAAUUGUACAGUAGUCUGCACAUUGGUUAGGCUGGGCUGGGUUAGACCCUCGG> hsa-mir-200a MI 0000737CCGGGCCCCUGUGAGCAUCUUACCGGACAGUGCUGGAUUUCCCAGCUUGACUCUAACACUGUCUGGUAACGAUGUUCAAAGGUGACCCGC> hsa-mir-200b MI0000342CCAGCUCGGGCAGCCGUGGCCAUCUUACUGGGCAGCAUUGGAUGGAGUCAGGUCUCUAAUACUGCCUGGUAAUGAUGACGGCGGAGCCCUGCACG> hsa-mir-200c MI0000650CCCUCGUCUUACCCAGCAGUGUUUGGGUGCGGUUGGGAGUCUCUAAUACUGCCGGGUAAUGAUGGAGG> hsa-mir-202 MI0003130CGCCUCAGAGCCGCCCGCCGUUCCUUUUUCCUAUGCAUAUACUUCUUUGAGGAUCUGGCCUAAAGAGGUAUAGGGCAUGGGAAAACGGGGCGGUCGGGUCCUCCCCAGCG> hsa-mir-203 MI0000283GUGUUGGGGACUCGCGCGCUGGGUCCAGUGGUUCUUAACAGUUCAACAGUUCUGUAGCGCAAUUGUGAAAUGUUUAGGACCACUAGACCCGGCGGGCGCGGCGACAGCGA> hsa-mir-204 MI0000284GGCUACAGUCUUUCUUCAUGUGACUCGUGGACUUCCCUUUGUCAUCCUAUGCCUGAGAAUAUAUGAAGGAGGCUGGGAAGGCAAAGGGACGUUCAAUUGUCAUCACUGGC> hsa-mir-205 MI0000285AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCACCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGUUCAGGAGGCAUGGAGCUGACA> hsa-mir-206 MI0000490UGCUUCCCGAGGCCACAUGCUUCUUUAUAUCCCCAUAUGGAUUACUUUGCUAUGGAAUGUAAGGAAGUGUGUGGUUUCGGCAAGUG > hsa-mir-208a MI0000251UGACGGGCGAGCUUUGGCCCGGGUUAUACCUGAU GCUCACGUAUAAGACGAGCAAAAAGCUUGUUGGUCA> hsa-mir-208b MI0005570CCUCUCAGGGAAGCUUUUUGCUCGAAUUAUGUUUCUGAUCCGAAUAUAAGACGAACAAAAGGUUUGUCUGAGGGCAG> hsa-mir-210 MI0000286ACCCGGCAGUGCCUCCAGGCGCAGGGCAGCCCCUGCCCACCGCACACUGCGCUGCCCCAGACCCACUGUGCGUGUGACAGCGGCUGAUCUGUGCCUGGGCAGCGCGACCC> hsa-mir-211 MI0000287UCACCUGGCCAUGUGACUUGUGGGCUUCCCUUUGUCAUCCUUCGCCUAGGGCUCUGAGCAGGGCAGGGACAGCAAAGGGGUGCUCAGUUGUCACUUCCCACAGCACGGAG> hsa-mir-212 MI0000288CGGGGCACCCCGCCCGGACAGCGCGCCGGCACCUUGGCUCUAGACUGCUUACUGCCCGGGCCGCCCUCAGUAACAGUCUCCAGUCACGGCCACCGACGCCUGGCCCCGCC> hsa-mir-214 MI0000290GGCCUGGCUGGACAGAGUUGUCAUGUGUCUGCCUGUCUACACUUGCUGUGCAGAACAUCCGCUCACCUGUACAGCAGGCACAGACAGGCAGUCACAUGACAACCCAGCCU> hsa-mir-215 MI0000291AUCAUUCAGAAAUGGUAUACAGGAAAAUGACCUAUGAAUUGACAGACAAUAUAGCUGAGUUUGUCUGUCAUUUCUUUAGGCCAAUAUUCUGUAUGACUGUGCUACUUCAA> hsa-mir-216a MI0000292GAUGGCUGUGAGUUGGCUUAAUCUCAGCUGGCAACUGUGAGAUGUUCAUACAAUCCCUCACAGUGGUCUCUGGGAUUAUGCUAAACAGAGCAAUUUCCUAGCCCUCACGA > hsa-mir-216b MI0005569GCAGACUGGAAAAUCUCUGCAGGCAAAU
GUGAUGUCACUGAGGAAAUCACACACUUACCCGUAGAGAUUCUACAGUCUGACA> hsa-mir-217 MI0000293AGUAUAAUUAUUACAUAGUUUUUGAUGUCGCAGAUACUGCAUCAGGAACUGAUUGGAUAAGAAUCAGUCACCAUCAGUUCCUAAUGCAUUGCCUUCAGCAUCUAAACAAG> hsa-mir-218-1 MI0000294GUGAUAAUGUAGCGAGAUUUUCUGUUGUGCUUGAUCUAACCAUGUGGUUGCGAGGUAUGAGUAAAACAUGGUUCCGUCAAGCACCAUGGAACGUCACGCAGCUUUCUACA> hsa-mir-218-2 MI0000295GACCAGUCGCUGCGGGGCUUUCCUUUGUGCUUGAUCUAACCAUGUGGUGGAACGAUGGAAACGGAACAUGGUUCUGUCAAGCACCGCGGAAAGCACCGUGCUCUCCUGCA> hsa-mir-219-1 MI0000296CCGCCCCGGGCCGCGGCUCCUGAUUGUCCAAACGCAAUUCUCGAGUCUAUGGCUCCGGCCGAGAGUUGAGUCUGGACGUCCCGAGCCGCCGCCCCCAAACCUCGAGCGGG> hsa-mir-219-2 MI0000740ACUCAGGGGCUUCGCCACUGAUUGUCCAAACGCAAUUCUUGUACGAGUCUGCGGCCAACCGAGAAUUGUGGCUGGACAUCUGUGGCUGAGCUCCGGG> hsa-mir-221 MI0000298UGAACAUCCAGGUCUGGGGCAUGAACCUGGCAUACAAUGUAGAUUUCUGUGUUCGUUAGGCAACAGCUACAUUGUCUGCUGGGUUUCAGGCUACCUGGAAACAUGUUCUC> hsa-mir-222 MI0000299GCUGCUGGAAGGUGUAGGUACCCUCAAUGGCU CCUCCUGCAGUGCCACGCUCCGUGUAUUUGACAAGCUGAGUUGGACACUCCAUGUGGUAGAGUGUCAGUUUGUCAAAUACCCCAAGUGCGGCACAUGCUUACCAG> hsa-mir-224 MI0000301GGGCUUUCAAGUCACUAGUGGUUCCGUUUAGUAGAUGAUUGUGCAUUGUUUCAAAAUGGUGCCCUAGUGACUACAAAGCCC> hsa-mir-296 MI0000747AGGACCCUUCCAGAGGGCCCCCCCUCAAUCCUGUUGUGCCUAAUUCAGAGGGUUGGGUGGAGGCUCUCCUGAAGGGCUCU> hsa-mir-297 MI0005775UGUAUGUAUGUGUGCAUGUGCAUGUAUGUGUAUAUACAUAUAUAUGUAUUAUGUACUCAUAUAUCA> hsa-mir-298 MI0005523UCAGGUCUUCAGCAGAAGCAGGGAGGUUCUCCCAGUGGUUUUCCUUGACUGUGAGGAACUAGCCUGCUGCUUUGCUCAGGAGUGAGCU> hsa-mir-299 MI0000744AAGAAAUGGUUUACCGUCCCACAUACAUUUUGAAUAUGUAUGUGGGAUGGUAAACCGCUUCUU> hsa-mir-300 MI0005525UGCUACUUGAAGAGAGGUAAUCCUUCACGCAUUUGCUUUACUUGCAAUGAUUAUACAAGGGCAGACUCUCUCUGGGGAGCAAA> hsa-mir-301a MI0000745ACUGCUAACGAAUGCUCUGACUUUAUUGCACUACUGUACUUUACAGCUAGCAGUGCAAUAGUAUUGUCAAAGCAUCUGAAAGCAGG > hsa-mir-301b MI0005568GCCGCAGGUGCUCUGACGAGGUUGCACUACUGUGCUCUGAGAAGCAGUGCAAUGAUUGUCAAAGCAUCUGGGACCA> hsa-mir-302a MI0000738CCACCACUUAAACGUGGAUGUACUUGCUUUGAAACUAAAGAAGUAAGUGCUUC > Hsa-mir-302b MI0000772GCUCCCUUCAACUUUAACAUGGAAGUGCUUUCUGUGACUUUAAAAGUAAGUGCUUCCAUGUUUUAGUAGGAGU> hsa-mir-302c MI0000773CCUUUGCUUUAACAUGGGGGUACCUGCUGUGUGAAACAAAAGUAAGUGCUUCCAUGUUUCAGUGGAGG> hsa-mir-302d MI0000774CCUCUACUUUAACAUGGAGGCACUUGCUGUGACAUGACAAAAAUAAGUGCUUCCAUGUUUGAGUGUGG> hsa-mir-302e MI0006417UUGGGUAAGUGCUUCCAUGCUUCAGUUUCCUUACUGGUAAGAUGGAUGUAGUAAUAGCACCUACCUUAUAGA> hsa-mir-302f MI0006418UCUGUGUAAACCUGGCAAUUUUCACUUAAUUGCUUCCAUGUUUAUAAAAGA> hsa-mir-320a MI0000542GCUUCGCUCCCCUCCGCCUUCUCUUCCCGGUUCUUCCCGGAGUCGGGAAAAGCUGGGUUGAGAGGGCGAAAAAGGAUGAGGU> hsa-mir-320b-1 MI0003776AAUUAAUCCCUCUCUUUCUAGUUCUUCCUAGAGUGAGGAAAAGCUGGGUUGAGAGGGCAAACAAAUUAACUAAUUAAUU> hsa-mir-320b-2 MI0003839UGUUAUUUUUUGUCUUCUACCUAAGAAUUCUGUCUCUUAGGCUUUCUCUUCCCAGAUUUCCCAAAGUUGGGAAAAGCUGGGUUGAGAGGGCAAAAGGAAAAAAAAAGAAUUCUGUCUCUGACAUAAUUAGAUAGGGAA> hsa-mir-320c-1 MI0003778UUUGCAUUAAAAAUGAGGCCUUCUCUUCCCAGUUCUUCCCAGAGUCAGGAAAAGCUGGGUUGAGAGGGUAGAAAAAAAAUGAUGUAGG> hsa-mir-320c-2 MI0008191CUUCUCUUUCCAGUUCUUCCCAGAAUUGGGAAAAGCUGGGUUG AGAGGGU> hsa-mir-320d-1 MI0008190UUCUCGUCCCAGUUCUUCCCAAAGUUGAGAAAAGCUGGGUUGAGAGGA> hsa-mir-320d-2 MI0008192UUCUCUUCCCAGUUCUUCUUGGAGUCAGGAAAAGCUGGGUUGAGAGGA> hsa-mir-320e MI0014234GCCUUCUCUUCCCAGUUCUUCCUGGAGUCGGGGAAAAGCUGGGUUGAGAAGGU> hsa-mir-323a MI0000807UUGGUACUUGGAGAGAGGUGGUCCGUGGCGCGUUCGCUUUAUUUAUGGCGCACAUUACACGGUCGACCUCUUUGCAGUAUCUAAUC> hsa-mir-323b MI0014206UGGUACUCGGAGGGAGGUUGUCCGUGGUGAGUUCGCAUUAUUUAAUGAUGCCCAAUACACGGUCGACCUCUUUUCGGUAUCA> hsa-mir-324 MI0000813CUGACUAUGCCUCCCCGCAUCCCCUAGGGCAUUGGUGUAAAGCUGGAGACCCACUGCCCCAGGUGCUGCUGGGGGUUGUAGUC> hsa- mir-325 MI0000824AUACAGUGCUUGGUUCCUAGUAGGUGUCCAGUAAGUGUUUGUGACAUAAUUUGUUUAUUGAGGACCUCCUAUCAAUCAAGCACUGUGCUAGGCUCUGG> hsa-mir-326 MI0000808CUCAUCUGUCUGUUGGGCUGGAGGCAGGGCCUUUGUGAAGGCGGGUGGUGCUCAGAUCGCCUCUGGGCCCUUCCUCCAGCCCCGAGGCGGAUUCA> hsa-mir-328 MI0000804UGGAGUGGGGGGGCAGGAGGGGCUCAGGGAGAAAGUGCAUACAGCCCCUGGCCCUCUCUGCCCUUCCGUCCCCUG> hsa-mir-329-1 MI0001725GGUACCUGAAGAGAGGUUUUCUGGGUUUCUGUUUCUUUAAUGAGGACGAAACACACCUGGUUAACCUCUUUUCCAGUAUC> hsa-mir-329-2 MI0001726GUGGUACCUGAAGAGAGGUUUUCUGGGUUUCUGUUUCUUUAUUGAGGACGAAACACACCUGGUUAACCUCUUUUCCAGUAUCAA> hsa-mir-330 MI0000803CUUUGGCGAUCACUGCCUCUCUGGGCCUGUGUCUUAGGCUCUGCAAGAUCAACCGAGCAAAGCACACGGCCUGCAGAGAGGCAGCGCUCUGCCC> hsa-mir-331 MI0000812GAGUUUGGUUUUGUUUGGGUUUGUUCUAGGUAUGGUCCCAGGGAUCCCAGAUCAAACCAGGCCCCUGGGCCUAUCCUAGAACCAACCUAAGCUC> hsa-mir-335 MI0000816UGUUUUGAGCGGGGGUCAAGAGCAAUAACGAAAAAUGUUUGUCAUAAACCGUUUUUCAUUAUUGCUCCUGACCUCCUCUCAUUUGCUAUAUUCA> hsa-mir-337 MI0000806GUAGUCAGUAGUUGGGGGGUGGGAACGGCUUCAUACAGGAGUUGAUGCACAGUUAUCCAGCUCCUAUAUGAUGCCUUUCUUCAUCCCCUUCAA> hsa-mir-338 MI0000814UCUCCAACAAUAUCCUGGUGCUGAGUGAUGACUCAGGCGACUCCAGCAUCAGUGAUUUUGUUGAAGA> hsa-mir-339 MI0000815CGGGGCGGCCGCUCUCCCUGUCCUCCAGGAGCUCACGUGUGCCUGCCUGUGAGCGCCUCGACGACAGAGCCGGCGCCUGCCCCAGUGUCUGCGC> hsa-mir-340 MI0000802UUGUACCUGGUGUGAUUAUAAAGCAAUGAGACUGAUUGUCAUAUGUCGUUUGUGGGAUCCGUCUCAGUUACUUUAUAGCCAUACCUGGUAUCUUA > hsa-mir-342 MI0000805GAAACUGGGCUCAAGGUGAGGGGUGCUAUCUGAUUGAGGGACAUGGUUAAUGGAAUUGUCACACAGAAAUCGCACCCGUCUCCUUGGCCUACUUA > Hsa-mir-345 MI0000825ACCCAAACCCUAGGUCUGCUGACUCCUAGUCCAGGGCUCGUGAUGGCUGGUGGGCCCUGAACGAGGGGUCUGGAGGCCUGGGUUUGAAUAUCGACAGC> hsa-mir-346 MI0000826GGUCUCUGUGUUGGGCGUCUGUCUGCCCGCAUGCCUGCCUCUCUGUUGCUCUGAAGGAGGCAGGGGCUGGGCCUGCAGCUGCCUGGGCAGAGCGG> hsa-mir-361 MI0000760GGAGCUUAUCAGAAUCUCCAGGGGUACUUUAUAAUUUCAAAAAGUCCCCCAGGUGUGAUUCUGAUUUGCUUC> hsa-mir-362 MI0000762CUUGAAUCCUUGGAACCUAGGUGUGAGUGCUAUUUCAGUGCAACACACCUAUUCAAGGAUUCAAA> hsa-mir-363 MI0000764UGUUGUCGGGUGGAUCACGAUGCAAUUUUGAUGAGUAUCAUAGGAGAAAAAUUGCACGGUAUCCAUCUGUAAACC> hsa-mir-365a MI0000767ACCGCAGGGAAAAUGAGGGACUUUUGGGGGCAGAUGUGUUUCCAUUCCACUAUCAUAAUGCCCCUAAAAAUCCUUAUUGCUCUUGCA> hsa-mir-365b MI0000769AGAGUGUUCAAGGACAGCAAGAAAAAUGAGGGACUUUCAGGGGCAGCUGUGUUUUCUGACUCAGUCAUAAUGCCCCUAAAAAUCCUUAUUGUUCUUGCAGUGUGCAUCGGG> hsa-mir-367 MI0000775CCAUUACUGUUGCUAAUAUGCAACUCUGUUGAAUAUAAAUUGGAAUUGCACUUUAGCAAUGGUGAUGG> hsa-mir-369 MI0000777UUGAAGGGAGAUCGACCGUGUUAUAUUCGCUUAUUGACUUCGAAUAAUACAUGGUUG GCUCACGAGUGCCUGCUGGGGUGGAACCUGGUCUGUCU> hsa-mir-371a MI0000779GUGGCACUCAAACUGUGGGGGCACUUUCUGCUCUCUGGUGAAAGUGCCGCCAUCUUUUGAGUGUUAC> hsa-mir-371b MI0017393GGUAACACUCAAAAGAUGGCGGCACUUUCACCAGAGAGCAGAAAGUGCCCCCACAGUUUGAGUGCC> hsa-mir-372 MI0000780GUGGGCCUCAAAUGUGGAGCACUAUUCUGAUGUCCAAGUGGAAAGUGCUGCGACAUUUGAGCGUCAC> hsa-mir-373 MI0000781GGGAUACUCAAAAUGGGGGCGCUUUCCUUUUUGUCUGUACUGGGAAGUGCUUCGAUUUUGGGGUGUCCC> hsa-mir-374a MI0000782UACAUCGGCCAUUAUAAUACAACCUGAUAAGUGUUAUAGCACUUAUCAGAUUGUAUUGUAAUUGUCUGUGUA> hsa-mir-374b MI0005566ACUCGGAUGGAUAUAAUACAACCUGCUAAGUGUCCUAGCACUUAGCAGGUUGUAUUAUCAUUGUCCGUGUCU> hsa-mir-374c MI0016684ACACGGACAAUGAUAAUACAACCUGCUAAGUGCUAGGACACUUAGCAGGUUGUAUUAUAUCCAUCCGAGU > hsa-mir-375 MI0000783CCCCGCGACGAGCCCCUCGCACAAACCGGACCUGAGCGUUUUGUUCGUUCGGCUCGCGUGAGGC> hsa-mir-376a-1 MI0000784UAAAAGGUAGAUUCUCCUUCUAUGAGUACAUUAUUUAUGAUUAAUCAUAGAGGAAAAUCCACGUUUUC> hsa-mir-376a-2 MI0003529GGUAUUUAAAAGGUAGAUUUUCCUUCUAUGGUUACGUGUUUGAUGGUUAAUCAUAGAGGAAAAUCCACGUUUUCAGUAUC> hsa-mir-376b MI0002466CAGUCCUUCUUUG GUAUUUAAAACGUGGAUAUUCCUUCUAUGUUUACGUGAUUCCUGGUUAAUCAUAGAGGAAAAUCCAUGUUUUCAGUAUCAAAUGCUG> hsa-mir-376c MI0000776AAAAGGUGGAUAUUCCUUCUAUGUUUAUGUUAUUUAUGGUUAAACAUAGAGGAAAUUCCACGUUUU> hsa-mir-377 MI0000785UUGAGCAGAGGUUGCCCUUGGUGAAUUCGCUUUAUUUAUGUUGAAUCACACAAAGGCAACUUUUGUUUG> hsa-mir-378a MI0000786AGGGCUCCUGACUCCAGGUCCUGUGUGUUACCUAGAAAUAGCACUGGACUUGGAGUCAGAAGGCCU> hsa-mir-378b MI0014154GGUCAUUGAGUCUUCAAGGCUAGUGGAAAGAGCACUGGACUUGGAGGCAGAAAGACC> hsa-mir-378c MI0015825GGAGGCCAUCACUGGACUUGGAGUCAGAAGAGUGGAGUCGGGUCAGACUUCAACUCUGACUUUGAAGGUGGUGAGUGCCUC> hsa-mir-378d-1 MI0016749ACUGUUUCUGUCCUUGUUCUUGUUGUUAUUACUGGACUUGGAGUCAGAAACAGG> hsa-mir- 378d-2 MI0003840GAAUGGUUACAAGGAGAGAACACUGGACUUGGAGUCAGAAAACUUUCAUCCAAGUCAUUCCCUGCUCUAAGUCCCAUUUCUGUUCCAUGAGAUUGUUU> hsa-mir-378e MI0016750CUGACUCCAGUGUCCAGGCCAGGGGCAGACAGUGGACAGAGAACAGUGCCCAAGACCACUGGACUUGGAGUCAGGACAU> hsa-mir-378f MI0016756GUCAGGUCCUGGACUCCCAUAGUUUUCAGGCUGCUAAACAACAGAACGAGCACUGGACUUGGAGCCAGAAGUCUUGGG> hsa-mir-378g MI0016761CACUGGGCUUGGAGUCAGAAGACCUGGCUCCA GCCCAGCUC> hsa-mir-378h MI0016808ACAGGAACACUGGACUUGGUGUCAGAUGGGAUGAGCCCUGGCUCUGUUUCCUAGCAGCAAUCUGAUCUUGAGCUAGUCACUGG> hsa-mir-378i MI0016902GGGAGCACUGGACUAGGAGUCAGAAGGUGGAGUUCUGGGUGCUGUUUUCCCACUCUUGGGCCCUGGGCAUGUUCUG> hsa-mir-379 MI0000787AGAGAUGGUAGACUAUGGAACGUAGGCGUUAUGAUUUCUGACCUAUGUAACAUGGUCCACUAACUCU> hsa-mir-380 MI0000788AAGAUGGUUGACCAUAGAACAUGCGCUAUCUCUGUGUCGUAUGUAAUAUGGUCCACAUCUU> hsa-mir-381 MI0000789UACUUAAAGCGAGGUUGCCCUUUGUAUAUUCGGUUUAUUGACAUGGAAUAUACAAGGGCAAGCUCUCUGUGAGUA> hsa-mir-382 MI0000790UACUUGAAGAGAAGUUGUUCGUGGUGGAUUCGCUUUACUUAUGACGAAUCAUUCACGGACAACACUUUUUUCAGUA> hsa-mir-383 MI0000791CUCCUCAGAUCAGAAGGUGAUUGUGGCUUUGGGUGGAUAUUAAUCAGCCACAGCACUGCCUGGUCAGAAAGAG > hsa-mir-384 MI0001145UGUUAAAUCAGGAAUUUUAAACAAUUCCUAGACAAUAUGUAUAAUGUUCAUAAGUCAUUCCUAGAAAUUGUUCAUAAUGCCUGUAACA> hsa-mir-409 MI0001735UGGUACUCGGGGAGAGGUUACCCGAGCAACUUUGCAUCUGGACGACGAAUGUUGCUCGGUGAACCCCUUUUCGGUAUCA> hsa-mir-410 MI0002465GGUACCUGAGAAGAGGUUGUCUGUGAUGAGUUCGCUUUUAUUAAUGACGAAUAUAACACAGAUGGCCUGUUUUCAGUACC> hsa-mir-41 1 MI0003675UGGUACUUGGAGAGAUAGUAGACCGUAUAGCGUACGCUUUAUCUGUGACGUAUGUAACACGGUCCACUAACCCUCAGUAUCAAAUCCAUCCCCGAG> hsa-mir-412 MI0002464CUGGGGUACGGGGAUGGAUGGUCGACCAGUUGGAAAGUAAUUGUUUCUAAUGUACUUCACCUGGUCCACUAGCCGUCCGUAUCCGCUGCAG> hsa-mir-421 MI0003685GCACAUUGUAGGCCUCAUUAAAUGUUUGUUGAAUGAAAAAAUGAAUCAUCAACAGACAUUAAUUGGGCGCCUGCUCUGUGAUCUC> hsa-mir-422a MI0001444GAGAGAAGCACUGGACUUAGGGUCAGAAGGCCUGAGUCUCUCUGCUGCAGAUGGGCUCUCUGUCCCUGAGCCAAGCUUUGUCCUCCCUGG> hsa-mir-423 MI0001445AUAAAGGAAGUUAGGCUGAGGGGCAGAGAGCGAGACUUUUCUAUUUUCCAAAAGCUCGGUCUGAGGCCCCUCAGUCUUGCUUCCUAACCCGCGC> hsa-mir-424 MI0001446CGAGGGGAUACAGCAGCAAUUCAUGUUUUGAAGUGUUCUAAAUGGUUCAAAACGUGAGGCGCUGCUAUACCCCCUCGUGGGGAAGGUAGAAGGUGGGG> hsa-mir-425 MI0001448GAAAGCGCUUUGGAAUGACACGAUCACUCCCGUUGAGUGGGCACCCGAGAAGCCAUCGGGAAUGUCGUGUCCGCCCAGUGCUCUUUC> hsa-mir-429 MI0001641CGCCGGCCGAUGGGCGUCUUACCAGACAUGGUUAGACCUGGCCCUCUGUCUAAUACUGUCUGGUAAAACCGUCCAUCCGCUGC> hsa-mir-431 MI0001721UCCUGCUUGUCCUGCGAGGUGUCUUGCAGGCCGUCAUGCAG
GCCACACUGACGGUAACGUUGCAGGUCGUCUUGCAGGGCUUCUCGCAAGACGACAUCCUCAUCACCAACGACG> hsa-mir-432 MI0003133UGACUCCUCCAGGUCUUGGAGUAGGUCAUUGGGUGGAUCCUCUAUUUCCUUACGUGGGCCACUGGAUGGCUCCUCCAUGUCUUGGAGUAGAUCA> hsa-mir-433 MI0001723CCGGGGAGAAGUACGGUGAGCCUGUCAUUAUUCAGAGAGGCUAGAUCCUCUGUGUUGAGAAGGAUCAUGAUGGGCUCCUCGGUGUUCUCCAGG> hsa-mir-448 MI0001637GCCGGGAGGUUGAACAUCCUGCAUAGUGCUGCCAGGAAAUCCCUAUUUCAUAUAAGAGGGGGCUGGCUGGUUGCAUAUGUAGGAUGUCCCAUCUCCCAGCCCACUUCGUCA> hsa-mir-449a MI0001648CUGUGUGUGAUGAGCUGGCAGUGUAUUGUUAGCUGGUUGAAUAUGUGAAUGGCAUCGGCUAACAUGCAACUGCUGUCUUAUUGCAUAUACA> hsa-mir-449b MI0003673UGACCUGAAUCAGGUAGGCAGUGUAUUGUUAGCUGGCUGCUUGGGUCAAGUCAGCAGCCACAACUACCCUGCCACUUGCUUCUGGAUAAAUUCUUCU> hsa-mir-449c MI0003823GCUGGGAUGUGUCAGGUAGGCAGUGUAUUGCUAGCGGCUGUUAAUGAUUUUAACAGUUGCUAGUUGCACUCCUCUCUGUUGCAUUCAGAAGC> hsa-mir-450a- 1 MI0001652AAACGAUACUAAACUGUUUUGCGAUGUGUUCCUAAUAUGCACUAUAAAUAUAUUGGGAACAUUUUGCAUGUAUAGUUUUGUAUCAAUAUA> hsa-mir-450a-2 MI0003187CCAAAGAAAGAUGCUAAACUAUUUUGCGAUGUUCCUAAUGUA AUUCAUAGUUUUGUAUCAAUAAUAUGG> hsa-mir-450b MI0005531GCAGAAUUAUUUUUGCAAUAUGUUCCUGAAUAUGUAAUAUAAGUGUAUUGGGAUCAUUUUGCAUCCAUAGUUUUGUAU> hsa-mir-451a MI0001729CUUGGGAAUGGCAAGGAAACCGUUACCAUUACUGAGUUUAGUAAUGGUAAUGGUUCUCUUGCUAUACCCAGA> hsa-mir-451b MI0017360UGGGUAUAGCAAGAGAACCAUUACCAUUACUAAACUCAGUAAUGGUAACGGUUUCCUUGCCAUUCCCA> hsa-mir-452 MI0001733GCUAAGCACUUACAACUGUUUGCAGAGGAAACUGAGACUUUGUAACUAUGUCUCAGUCUCAUCUGCAAAGAAGUAAGUGCUUUGC> hsa-mir-454 MI0003820UCUGUUUAUCACCAGAUCCUAGAACCCUAUCAAUAUUGUCUCUGCUGUGUAAAUAGUUCUGAGUAGUGCAAUAUUGCUUAUAGGGUUUUGGUGUUUGGAAAGAACAAUGGGCAGG> hsa-mir-455 MI0003513UCCCUGGCGUGAGGGUAUGUGCCUUUGGACUACAUCGUGGAAGCCAGCACCAUGCAGUCCAUGGGCAUAUACACUUGCCUCAAGGCCUAUGUCAUC> hsa-mir-466 MI0014157GUGUGUGUAUAUGUGUGUUGCAUGUGUGUAUAUGUGUGUAUAUAUGUACACAUACACAUACACGCAACACACAUAUAUACAUGC > hsa-mir-483 MI0002467GAGGGGGAAGACGGGAGGAAAGAAGGGAGGUUCCAUCACGCCUCCUCACUCCUCUCCUCGUCUUCUCCUCUC> hsa-mir-484 MI0002468AGCCUCGUCAGGCUCAGUCCCCUCCCGAUAAACCCCUAAAUAGGGACUUUCCGGG 469ACUUGGAGAGAGGCUGGCCGUGAUGAAUUCGAUUCAUCAAAGCGAGUCAUACACGGCUCUCCUCUCUUUUAGU> hsa-mir-486 MI0002470GCAUCCUGUACUGAGCUGCCCCGAGGCCCUUCAUGCUGCCCAGCUCGGGGCAGCUCAGUACAGGAUAC> hsa-mir-487a MI0002471GGUACUUGAAGAGUGGUUAUCCCUGCUGUGUUCGCUUAAUUUAUGACGAAUCAUACAGGGACAUCCAGUUUUUCAGUAUC> hsa-mir-487b MI0003530UUGGUACUUGGAGAGUGGUUAUCCCUGUCCUGUUCGUUUUGCUCAUGUCGAAUCGUACAGGGUCAUCCACUUUUUCAGUAUCAA> hsa-mir-488 MI0003123GAGAAUCAUCUCUCCCAGAUAAUGGCACUCUCAAACAAGUUUCCAAAUUGUUUGAAAGGCUAUUUCUUGGUCAGAUGACUCUC> hsa-mir-489 MI0003124GUGGCAGCUUGGUGGUCGUAUGUGUGACGCCAUUUACUUGAACCUUUAGGAGUGACAUCACAUAUACGGCAGCUAAACUGCUAC> hsa-mir-490 MI0003125UGGAGGCCUUGCUGGUUUGGAAAGUUCAUUGUUCGACACCAUGGAUCUCCAGGUGGGUCAAGUUUAGAGAUGCACCAACCUGGAGGACUCCAUGCUGUUGAGCUGUUCACAAGCAGCGGACACUUCCA> hsa-mir-491 MI0003126UUGACUUAGCUGGGUAGUGGGGAACCCUUCCAUGAGGAGUAGAACACUCCUUAUGCAAGAUUCCCUUCUACCUGGCUGGGUUGG > hsa-mir-492 MI0003131CAACUACAGCCACUACUACAGGACCAUCGAGGACCUGCGGGACAAGAUUCUUGGUGCCACCAUUGAGAACGCCAGGAUUGUCCUGCAGAUCAACAAUGCUCAACUGGCUGCAGAUG> hsa-mir-493 MI0003 132CUGGCCUCCAGGGCUUUGUACAUGGUAGGCUUUCAUUCAUUCGUUUGCACAUUCGGUGAAGGUCUACUGUGUGCCAGGCCCUGUGCCAG> hsa-mir-494 MI0003134GAUACUCGAAGGAGAGGUUGUCCGUGUUGUCUUCUCUUUAUUUAUGAUGAAACAUACACGGGAAACCUCUUUUUUAGUAUC> hsa-mir-495 MI0003135UGGUACCUGAAAAGAAGUUGCCCAUGUUAUUUUCGCUUUAUAUGUGACGAAACAAACAUGGUGCACUUCUUUUUCGGUAUCA> hsa-mir-496 MI0003136CCCAAGUCAGGUACUCGAAUGGAGGUUGUCCAUGGUGUGUUCAUUUUAUUUAUGAUGAGUAUUACAUGGCCAAUCUCCUUUCGGUACUCAAUUCUUCUUGGG> hsa-mir-497 MI0003138CCACCCCGGUCCUGCUCCCGCCCCAGCAGCACACUGUGGUUUGUACGGCACUGUGGCCACGUCCAAACCACACUGUGGUGUUAGAGCGAGGGUGGGGGAGGCACCGCCGAGG> hsa-mir-498 MI0003142AACCCUCCUUGGGAAGUGAAGCUCAGGCUGUGAUUUCAAGCCAGGGGGCGUUUUUCUAUAACUGGAUGAAAAGCACCUCCAGAGCUUGAAGCUCACAGUUUGAGAGCAAUCGUCUAAGGAAGUU> hsa-mir-499a MI0003183GCCCUGUCCCCUGUGCCUUGGGCGGGCGGCUGUUAAGACUUGCAGUGAUGUUUAACUCCUCUCCACGUGAACAUCACAGCAAGUCUGUGCUGCUUCCCGUCCCUACGCUGCCUGGGCAGGGU> hsa-mir-499b MI0017396GGAAGCAGCACAGACUUGCUGUGAUGUUCACGUGGAGAGGAGUUAAACAUCACUGCAAGUCUUAACAGCCGCC > hsa-mir-500a MI0003184GCUCCCCCUCUCUAAUCCUUGCUACCUGGGUGA GAGUGCUGUCUGAAUGCAAUGCACCUGGGCAAGGAUUCUGAGAGCGAGAGC> hsa-mir-500b MI0015903CCCCCUCUCUAAUCCUUGCUACCUGGGUGAGAGUGCUUUCUGAAUGCAGUGCACCCAGGCAAGGAUUCUGCAAGGGGGA> hsa-mir-501 MI0003185GCUCUUCCUCUCUAAUCCUUUGUCCCUGGGUGAGAGUGCUUUCUGAAUGCAAUGCACCCGGGCAAGGAUUCUGAGAGGGUGAGC> hsa-mir-502 MI0003186UGCUCCCCCUCUCUAAUCCUUGCUAUCUGGGUGCUAGUGCUGGCUCAAUGCAAUGCACCUGGGCAAGGAUUCAGAGAGGGGGAGCU> hsa-mir-503 MI0003188UGCCCUAGCAGCGGGAACAGUUCUGCAGUGAGCGAUCGGUGCUCUGGGGUAUUGUUUCCGCUGCCAGGGUA> hsa-mir-504 MI0003189GCUGCUGUUGGGAGACCCUGGUCUGCACUCUAUCUGUAUUCUUACUGAAGGGAGUGCAGGGCAGGGUUUCCCAUACAGAGGGC> hsa-mir-505 MI0003190GAUGCACCCAGUGGGGGAGCCAGGAAGUAUUGAUGUUUCUGCCAGUUUAGCGUCAACACUUGCUGGUUUCCUCUCUGGAGCAUC> hsa-mir-506 MI0003193GCCACCACCAUCAGCCAUACUAUGUGUAGUGCCUUAUUCAGGAAGGUGUUACUUAAUAGAUUAAUAUUUGUAAGGCACCCUUCUGAGUAGAGUAAUGUGCAACAUGGACAACAUUUGUGGUGGC > hsa-mir-507 MI0003194GUGCUGUGUGUAGUGCUUCACUUCAAGAAGUGCCAUGCAUGUGUCUAGAAAUAUGUUUGCACCUUUUGGAGUGAAAUAAUGCACAACAGAUAC> hsa-mir-508 MI0003195CCACCUUCCUGAGUGUAGUGCCCUACUCCAGAGG AUGUAAACUAAAACAUGAUUGUAGCCUUUUGGAGUAGAGUAAUACACAUCACGUAACGCAUAUUUGGUGG> hsa-mir-509-1 MI0003196CAUGCUGUGUGUGGUACCCUACUGCAGACAGUGGCAAUCAUGUAUAAUUAAAAAUGAUUGGUACGUCUGUGGGUAGAGUACUGCAUGACACAUG> hsa-mir-509-2 MI0005530CAUGCUGUGUGUGGUACCCUACUGCAGACAGUGGCAAUCAUGUAUAAUUAAAAAUGAUUGGUACGUCUGUGGGUAGAGUACUGCAUGACAC> hsa-mir-509-3 MI0005717GUGGUACCCUACUGCAGACGUGGCAAUCAUGUAUAAUUAAAAAUGAUUGGUACGUCUGUGGGUAGAGUACUGCAU> hsa-mir-510 MI0003197GUGGUGUCCUACUCAGGAGAGUGGCAAUCACAUGUAAUUAGGUGUGAUUGAAACCUCUAAGAGUGGAGUAACAC> hsa-mir-511-1 MI0003127CAAUAGACACCCAUCGUGUCUUUUGCUCUGCAGUCAGUAAAUAUUUUUUUGUGAAUGUGUAGCAAAAGACAGAAUGGUGGUCCAUUG> hsa-mir-511 -2 MI0003128CAAUAGACACCCAUCGUGUCUUUUGCUCUGCAGUCAGUAAAUAUUUUUUUGUGAAUGUGUAGCAAAAGACAGAAUGGUGGUCCAUUG> hsa-mir-512-1 MI0003140UCUCAGUCUGUGGCACUCAGCCUUGAGGGCACUUUCUGGUGCCAGAAUGAAAGUGCUGUCAUAGCUGAGGUCCAAUGACUGAGG> hsa-mir-512-2 MI0003141GGUACUUCUCAGUCUGUGGCACUCAGCCUUGAGGGCACUUUCUGGUGCCAGAAUGAAAGUGCUGUCAUAGCUGAGGUCCAAUGACUGAGGCGAGCACC> hsa-mir-513a-1 MI0003191GGGAUGCCACAUUCAGCCAUUCAGC GUACAGUGCCUUUCACAGGGAGGUGUCAUUUAUGUGAACUAAAAUAUAAAUUUCACCUUUCUGAGAAGGGUAAUGUACAGCAUGCACUGCAUAUGUGGUGUCCC> hsa-mir-513a-2 MI0003192GGAUGCCACAUUCAGCCAUUCAGUGUGCAGUGCCUUUCACAGGGAGGUGUCAUUUAUGUGAACUAAAAUAUAAAUUUCACCUUUCUGAGAAGGGUAAUGUACAGCAUGCACUGCAUAUGUGGUGUCC> hsa-mir-513b MI0006648GUGUACAGUGCCUUUCACAAGGAGGUGUCAUUUAUGUGAACUAAAAUAUAAAUGUCACCUUUUUGAGAGGAGUAAUGUACAGCA> hsa-mir-513c MI0006649GCGUACAGUGCCUUUCUCAAGGAGGUGUCGUUUAUGUGAACUAAAAUAUAAAUUUCACCUUUCUGAGAAGAGUAAUGUACAGCA> hsa-mir-514a-1 MI0003198AACAUGUUGUCUGUGGUACCCUACUCUGGAGAGUGACAAUCAUGUAUAAUUAAAUUUGAUUGACACUUCUGUGAGUAGAGUAACGCAUGACACGUACG> hsa-mir-514a-2 MI0003199GUUGUCUGUGGUACCCUACUCUGGAGAGUGACAAUCAUGUAUAACUAAAUUUGAUUGACACUUCUGUGAGUAGAGUAACGCAUGACAC> hsa-mir-514a-3 MI0003200GUUGUCUGUGGUACCCUACUCUGGAGAGUGACAAUCAUGUAUAACUAAAUUUGAUUGACACUUCUGUGAGUAGAGUAACGCAUGACAC> hsa-mir-514b MI0014251CAUGUGGUACUCUUCUCAAGAGGGAGGCAAUCAUGUGUAAUUAGAUAUGAG GCCUUCUUUUGGAGCGUUACUGUUUGAGA> hsa-mir-515-2 MI0003147UCUCAUGCAGUCAUUCUCCAAAAGAAAGCACUUUCUGUUGUCUGAAAGCAGAGUGCCUUCUUUUGGAGCGUUACUGUUUGAGA> hsa-mir-516a-1 MI0003180UCUCAGGCUGUGACCUUCUCGAGGAAAGAAGCACUUUCUGUUGUCUGAAAGAAAAGAAAGUGCUUCCUUUCAGAGGGUUACGGUUUGAGA> hsa-mir-516a-2 MI0003181UCUCAGGUUGUGACCUUCUCGAGGAAAGAAGCACUUUCUGUUGUCUGAAAGAAAAGAAAGUGCUUCCUUUCAGAGGGUUACGGUUUGAGA> hsa-mir-516b-1 MI0003172UCUCAGGCUGUGACCAUCUGGAGGUAAGAAGCACUUUCUGUUUUGUGAAAGAAAAGAAAGUGCUUCCUUUCAGAGGGUUACUCUUUGAGA> hsa-mir-516b-2 MI0003167UCUCAUGAUGUGACCAUCUGGAGGUAAGAAGCACUUUGUGUUUUGUGAAAGAAAGUGCUUCCUUUCAGAGGGUUACUCUUUGAGA> hsa-mir -517a MI0003161UCUCAGGCAGUGACCCUCUAGAUGGAAGCACUGUCUGUUGUAUAAAAGAAAAGAUCGUGCAUCCCUUUAGAGUGUUACUGUUUGAGA> hsa-mir-517b MI0003165GUGACCCUCUAGAUGGAAGCACUGUCUGUUGUCUAAGAAAAGAUCGUGCAUCCCUUUAGAGUGUUAC> hsa-mir-517c MI0003174GAAGAUCUCAGGCAGUGACCCUCUAGAUGGAAGCACUGUCUGUUGUCUAAGAAAAGAUCGUGCAUCCUUUUAGAGUGUUACUGUUUGAGAAAAUC> hsa-mir-518a-1 MI0003170UCUCAAGCUGUGACUGCAAAGGGAAGCCCUUUCUGUUGUCUGAAAGAAGAGAAAGCGCUUCCCUUU GCUGGAUUACGGUUUGAGA> hsa-mir-518a-2 MI0003173UCUCAAGCUGUGGGUCUGCAAAGGGAAGCCCUUUCUGUUGUCUAAAAGAAGAGAAAGCGCUUCCCUUUGCUGGAUUACGGUUUGAGA> hsa-mir-518b MI0003156UCAUGCUGUGGCCCUCCAGAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAACAAAGCGCUCCCCUUUAGAGGUUUACGGUUUGA> hsa-mir-518c MI0003159GCGAGAAGAUCUCAUGCUGUGACUCUCUGGAGGGAAGCACUUUCUGUUGUCUGAAAGAAAACAAAGCGCUUCUCUUUAGAGUGUUACGGUUUGAGAAAAGC> hsa-mir-518d MI0003171UCCCAUGCUGUGACCCUCUAGAGGGAAGCACUUUCUGUUGUCUGAAAGAAACCAAAGCGCUUCCCUUUGGAGCGUUACGGUUUGAGA> hsa-mir-518e MI0003169UCUCAGGCUGUGACCCUCUAGAGGGAAGCGCUUUCUGUUGGCUAAAAGAAAAGAAAGCGCUUCCCUUCAGAGUGUUAACGCUUUGAGA> hsa-mir-518f MI0003154UCUCAUGCUGUGACCCUCUAGAGGGAAGCACUUUCUCUUGUCUAAAAGAAAAGAAAGCGCUUCUCUUUAGAGGAUUACUCUUUGAGA> hsa-mir- 519a-1 MI0003178CUCAGGCUGUGACACUCUAGAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAGGAAAGUGCAUCCUUUUAGAGUGUUACUGUUUGAG> hsa-mir-519a-2 MI0003182UCUCAGGCUGUGUCCCUCUACAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAGGAAAGUGCAUCCUUUUAGAGUGUUACUGUUUGAGA> hsa-mir-519b MI0003151CAUGCUGUGACCCUCUAGAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAAGAAAGUGCAUCCUU UUAGAGGUUUACUGUUUG> hsa-mir-519c MI0003148UCUCAGCCUGUGACCCUCUAGAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAAGAAAGUGCAUCUUUUUAGAGGAUUACAGUUUGAGA> hsa-mir-519d MI0003162UCCCAUGCUGUGACCCUCCAAAGGGAAGCGCUUUCUGUUUGUUUUCUCUUAAACAAAGUGCCUCCCUUUAGAGUGUUACCGUUUGGGA> hsa-mir-519e MI0003145UCUCAUGCAGUCAUUCUCCAAAAGGGAGCACUUUCUGUUUGAAAGAAAACAAAGUGCCUCCUUUUAGAGUGUUACUGUUUGAGA> hsa-mir-520a MI0003149CUCAGGCUGUGACCCUCCAGAGGGAAGUACUUUCUGUUGUCUGAGAGAAAAGAAAGUGCUUCCCUUUGGACUGUUUCGGUUUGAG> hsa-mir-520b MI0003155CCCUCUACAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAAGAAAGUGCUUCCUUUUAGAGGG> hsa-mir-520c MI0003158UCUCAGGCUGUCGUCCUCUAGAGGGAAGCACUUUCUGUUGUCUGAAAGAAAAGAAAGUGCUUCCUUUUAGAGGGUUACCGUUUGAGA> hsa-mir-520d MI0003164UCUCAAGCUGUGAGUCUACAAAGGGAAGCCCUUUCUGUUGUCUAAAAGAAAAGAAAGUGCUUCUCUUUGGUGGGUUACGGUUUGAGA > hsa-mir-520e MI0003143UCUCCUGCUGUGACCCUCAAGAUGGAAGCAGUUUCUGUUGUCUGAAAGGAAAGAAAGUGCUUCCUUUUUGAGGGUUACUGUUUGAGA> hsa-mir-520f MI0003146UCUCAGGCUGUGACCCUCUAAAGGGAAGCU
A> hsa-mir-520g MI0003166UCCCAUGCUGUGACCCUCUAGAGGAAGCACUUUCUGUUUGUUGUCUGAGAAAAAACAAAGUGCUUCCCUUUAGAGUGUUACCGUUUGGGA> hsa-mir-520h MI0003175UCCCAUGCUGUGACCCUCUAGAGGAAGCACUUUCUGUUUGUUGUCUGAGAAAAAACAAAGUGCUUCCCUUUAGAGUUACUGUUUGGGA> hsa-mir-521-1 MI0003176UCUCAGGCUGUGACCCUCCAAAGGGAAGAACUUUCUGUUGUCUAAAAGAAAAGAACGCACUUCCCUUUAGAGUGUUACCGUGUGAGA> hsa-mir-521-2 MI0003163UCUCGGGCUGUGACUCUCCAAAGGGAAGAAUUUUCUCUUGUCUAAAAGAAAAGAACGCACUUCCCUUUAGAGUGUUACCGUGUGAGA> hsa-mir-522 MI0003177UCUCAGGCUGUGUCCCUCUAGAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAAGAAAAUGGUUCCCUUUAGAGUGUUACGCUUUGAGA> hsa-mir-523 MI0003153UCUCAUGCUGUGACCCUCUAGAGGGAAGCGCUUUCUGUUGUCUGAAAGAAAAGAACGCGCUUCCCUAUAGAGGGUUACCCUUUGAGA> hsa- mir-524 MI0003160UCUCAUGCUGUGACCCUACAAAGGGAAGCACUUUCUCUUGUCCAAAGGAAAAGAAGGCGCUUCCCUUUGGAGUGUUACGGUUUGAGA> hsa-mir-525 MI0003152CUCAAGCUGUGACUCUCCAGAGGGAUGCACUUUCUCUUAUGUGAAAAAAAAGAAGGCGCUUCCCUUUAGAGCGUUACGGUUUGGG> hsa-mir-526a-1 MI0003157CUCAGGCUGUGACCCUCUAGAGGGAAGCACUUUCUGUUGCUUGAAAGAAGAGAAAGCGCUUCCUUUUAGAGGAUUACUCUUUGAG> hsa-mir- 526a-2 MI0003168GUGACCCUCUAGAGGGAAGCACUUUCUGUUGAAAGAAAAGAACAUGCAUCCUUUCAGAGGGUUAC> hsa-mir-526b MI0003150UCAGGCUGUGACCCUCUUGAGGGAAGCACUUUCUGUUGUCUGAAAGAAGAGAAAGUGCUUCCUUUUAGAGGCUUACUGUCUGA> hsa-mir-527 MI0003179UCUCAAGCUGUGACUGCAAAGGGAAGCCCUUUCUGUUGUCUAAAAGAAAAGAAAGUGCUUCCCUUUGGUGAAUUACGGUUUGAGA> hsa-mir-532 MI0003205CGACUUGCUUUCUCUCCUCCAUGCCUUGAGUGUAGGACCGUUGGCAUCUUAAUUACCCUCCCACACCCAAGGCUUGCAAAAAAGCGAGCCU> hsa-mir-539 MI0003514AUACUUGAGGAGAAAUUAUCCUUGGUGUGUUCGCUUUAUUUAUGAUGAAUCAUACAAGGACAAUUUCUUUUUGAGUAU> hsa-mir-541 MI0005539ACGUCAGGGAAAGGAUUCUGCUGUCGGUCCCACUCCAAAGUUCACAGAAUGGGUGGUGGGCACAGAAUCUGGACUCUGCUUGUG> hsa-mir-542 MI0003686CAGAUCUCAGACAUCUCGGGGAUCAUCAUGUCACGAGAUACCAGUGUGCACUUGUGACAGAUUGAUAACUGAAAGGUCUGGGAGCCACUCAUCUUCA> hsa-mir -543 MI0005565UACUUAAUGAGAAGUUGCCCGUGUUUUUUCGCUUUAUUGUGACGAAACAUUCGCGGUGCACUUCUUUUUCAGUAUC> hsa-mir-544a MI0003515AUUUUCAUCCUAGGGAUCUUGUUAAAAAGCAGAUUCCUAG GUAGCUCUGAGACUAGACCUGAGGUUGUGCAUUUCUAACAAAGUGCC> hsa-mir-545 MI0003516CCCAGCCUGGCACAUUAGUAGGCCUCAGUAAAUGUUUAUUAGAUGAAUAAAUGAAUGACUCAUCAGCAAACAUUUAUUGUGUGCCUGCUAAAGUGAGCUCCACAGG> hsa-mir-548a-1 MI0003593UGCAGGGAGGUAUUAAGUUGGUGCAAAAGUAAUUGUGAUUUUUGCCAUUAAAAGUAACGACAAAACUGGCAAUUACUUUUGCACCAAACCUGGUAUU> hsa-mir-548a-2 MI0003598UGUGAUGUGUAUUAGGUUUGUGCAAAAGUAAUUGGGGUUUUUUGCCGUUAAAAGUAAUGGCAAAACUGGCAAUUACUUUUGCACCAAACUAAUAUAA> hsa-mir-548a-3 MI0003612CCUAGAAUGUUAUUAGGUCGGUGCAAAAGUAAUUGCGAGUUUUACCAUUACUUUCAAUGGCAAAACUGGCAAUUACUUUUGCACCAACGUAAUACUU> hsa-mir-548aa-1 MI0016689CUUUAUUAGUCUGGUGCAAAAGAAACUGUGGUUUUUGCCAUUACUUUUACAGGCAAAAACCACAAUUACUUUUGCACCAACCUAAUAUAACUUGUUU> hsa-mir-548aa -2 MI0016690UUUUAUUAGGUUGGUGCAAAAGAAACUGUGGUUUUUGCCAUUACUUUCAAUGGCAAAAACCACAAUUACUUUUGCACCAACCUAAAUCUUCCCUCUC> hsa-mir-548ab MI0016752AUGUUGGUGCAAAAGUAAUUGUGGAUUUUGCUAUUACUUGUAUUUAUUUGUAAUGCAAAACCCGCAAUUAGUUUUGCACCAACC> hsa-mir-548ac MI0016762GUAUUAGGUUGGUGCAAAAGUUAUUGUGGUUUUUGCUAUUUUUUUUUAAUGGCAAAAACCGGCAAUUACUUUUGCACUAAC CUAGUAG> hsa-mir-548ad MI0016770CUGUUAGGUUGGUGCAAAAGUAAUUGUGGUUUUUGAAAGUAACUUGGCGAAAACGACAAUGACUUUUGCACCAAUCUAAUAC> hsa-mir-548ae-1 MI0016779GCAGUUUUUGCCAUUAAGUUGCGGUUUUUGCCAUUAUAAUGGCAAAAACUGCAAUUACUUUCACACCUGC> hsa-mir-548ae-2 MI0016780UGUGCAAAAGUAAUUGUGGUUUUUGUCAUUUAAAAGUAAUGGCAAAAACUGCAAUUACUUUCACACC> hsa-mir-548ag-1 MI0016793GUGCAAAGGUAAUUGUGGUUUCUGCUUUUAAAGGUAAUGGCAAAUAUUACAUUUACUUUUGCACCA> hsa-mir-548ag-2 MI0016794UGCAAAGGUAAUUGUGGUUUCUGCCAUUGAAAGUAAAGGCAAGAACCUCAAUUACCUUUGCAGC> hsa-mir-548ah MI0016796AGGUUGGUGCAAAAGUGAUUGCAGUGUUUGCCAAUAAAAGUAAUGACAAAAACUGCAGUUACUUUUGCACCAGCCC> hsa-mir-548ai MI0016813GUAUUAGGUUGGUGCAAAGGUAAUUGCAGUUUUUCCCAUUUAAAAUAUGGAAAAAAAAAUCACAAUUACUUUUGCAUCAACCUAAUAA> hsa-mir-548aj-1 MI0016814AUUGGUGUAAAAGUAAUUGCAGGUUAUGCCAUUAAAAGUAAUGGUAAAAACUGCAAUUACUUUUACACUAAC> hsa-mir-548aj-2 MI0016815AAGGUAUUAGGUUGGUGCAAAAGUAAUUGCAGUUUUUGCUAUUACUUUUAAUGGUAAAAACUGCAAUUACUUUUACACCAACCUAAUAUUUA> hsa-mir-548ak MI0016840GUGCAAAAGUAACUGCGGUUUUUGAGAAGUAAUUGAAAACCGCAAUUACUUUUGCAG> hsa-mi r-548al MI0016851GGUCGGUGCAAAAGUAAUUGCUGUUUUUGCCAUUAAAAAUAAUGGCAUUAAAAGUAAUGGCAAAAACGGCAAUGACUUUUGUACCAAUCUAAUAUCU> hsa-mir-548am MI0016904AGUUGGUGCAAAAGUAAUUGCGGUUUUUGCCGUCGAAAAUAAUGGCAAAAACUGCAGUUACUUUUGUACCAAUG> hsa-mir-548an MI0016907CAUUAGGUUGGUGCAAAAGGCAUUGUGGUUUUUGCCUAUAAAAGUAAUGGCAAAAACCGCAAUUCCUUUUGCACCAACCUAAU> hsa-mir-548ao MI0017871AACUAUUCUUAGGUUGAUGCAGAAGUAACUACGGUUUUUGCAGUUGAAAGUAAUGGCAAAGACCGUGACUACUUUUGCAACAGCCUAAUAGUUUCU> hsa-mir-548ap MI0017875ACCAAUUCCUAGGUUGGUGCAAAAGUAAUUGCGGUCUUUGUCAUUAAAACCAAUAACAAAAACCACAAUUACUUUUUACUGACCUAAAGAUUAAUU> hsa-mir-548aq MI0019130GAAAGUAAUUGCUGUUUUUGCCAUUACUUUCAGUGGCAAAAACUGCAAUUACUUUUGC> hsa-mir-548ar MI0019131AAAAGUAAUUGCAGUUUUUGCUGUUGAACGUAGUGGUAAAACUGCAGUUAUUUUUGC> hsa-mir -548as MI0019132AAAAGUAAUUGCGGGUUUUGCCGUUGCUUUUAAUGGUAAAACCCACAAUUAUGUUUGU> hsa-mir-548at MI0019137AAAAGUUAUUGCGGUUUUGGCUGCCAAAAGAAAUGGCCAAAACCGCAGUAACUUUUGU> hsa-mir-548au GGAUUUGCCAUCACCUUUACCUUUAAUGGCAAAACUGCAGUUACUUUUGC> hsa-mir-548aw MI0019283UAGGUCGGUGCAAAAGUCAUCACGGUUUUUACCAUUAAAACCGCGAUGACUUUUGCAUCAACCUA> hsa-mir-548ax MI0019286GAUUGGUGCAGAAGUAAUUGCGGUUUUGCCAUGGAAAGUAAUGGCAAAAACCGUAAUUACUUUUGUACCAACC> hsa-mir-548b MI0003596CAGACUAUAUAUUUAGGUUGGCGCAAAAGUAAUUGUGGUUUUGGCCUUUAUUUUCAAUGGCAAGAACCUCAGUUGCUUUUGUGCCAACCUAAUACUU> hsa-mir-548c MI0003630CAUUGGCAUCUAUUAGGUUGGUGCAAAAGUAAUUGCGGUUUUUGCCAUUACUUUCAGUAGCAAAAAUCUCAAUUACUUUUGCACCAACUUAAUACUU> hsa-mir-548d-1 MI0003668AAACAAGUUAUAUUAGGUUGGUGCAAAAGUAAUUGUGGUUUUUGCCUGUAAAAGUAAUGGCAAAAACCACAGUUUCUUUUGCACCAGACUAAUAAAG> hsa-mir-548d-2 MI0003671GAGAGGGAAGAUUUAGGUUGGUGCAAAAGUAAUUGUGGUUUUUGCCAUUGAAAGUAAUGGCAAAAACCACAGUUUCUUUUGCACCAACCUAAUAAAA> hsa- mir-548e MI0006344UUAUUAGGUUGGUACAAAGCAAUCGCGGUUUUUGCUAUUACUUUUAAAGGCAAAAACUGAGACUACUUUUGCACCAACCUGAUAGAA> hsa-mir-548f-1 MI0006374AUUAGGUUGGUGCAAAAGUAAUCACAGUU UUUUUAUCAUUACUUUUAAUGGCAAAAACUGUAAUUACUUUUGCACCAACCUAAUAUUUUAGU> hsa-mir-548f-3 MI0006376AUUAGGUUGGUGCAAACCUAAUUGCAAUUUUUGCAGUUUUUUUAAGUAAUUGCAAAAACUGUAAUUACUUUUGCACCAACCUAAUAC> hsa-mir-548f-4 MI0006377GAGUUCUAACGUAUUAGGUUGGUGCAAAAGUAAUAGUGGUUUUUGCCAUUAAAAGUAAUGACAAAAACUGUAAUUACUUUUGGAACAAUAUUAAUAGAAUUUCAG> hsa-mir-548f-5 MI0006378UAUUAGGUUGCUGCAAAAGUAAUCAUGUUUUUUUCCAUUGUAAGUAAUGGGAAAAACUGUAAUUACUUUUGUACCAACCUAAUAGC> hsa-mir-548g MI0006395AGUUAUUAGAUUAGUGCAAAAGUAAUUGCAGUUUUUGCAUUACGUUCUAUGGCAAAACUGUAAUUACUUUUGUACCAACAUAAUACUUC> hsa-mir-548h-1 MI0006411UCUGUCCAUUAGGUGGGUGCAAAAGUAAUCGCGGUUUUUGUCAUUACUUUUAAUGGUAAAAACUGGAAUUACUUUUGCACUGACCUAAUAUUAAGCCAGAUA> hsa-mir-548h -2 MI0006412GUAUUAGGUUGGUGCAAAAGUAAUCGCGGUUUUUGUCAUUACUUUCAAUGGCAAACACCACAAUUACUUUUGCACCAACCUAAUAUAA> hsa-mir-548h-3 MI0006413UCUGAUUCUGCAUGUAUUAGGUUGGUGCAAAAGUAAUCGCGGUUUUUGUCAUUGAAAGUAAUAGCAAAAACUGCAAUUACUUUUGCACCAACCUAAAAGUAGUCACUGUCUUCAGAUA> hsa-mir-548h-4 MI0006414GCUAUUAGGUUGGUGCAAAAGUAAUCGCGGUUUUUGUCAUUACUUUAAUUACUUUACGUUUCAU UAAUGACAAAAACCGCAAUUACUUUUGCACCAACCUAAUACUUGCUA> hsa-mir-548h-5 MI0016751ACAAAAGUAAUCGCGGUUUUUGUCAUUACUUUUAACUGUAAAAACCACGGUUGCUUUUGC> hsa-mir-548i-1 MI0006421CAGAUGGCUCUGAAGUUUGCACCCUAUUAGGUUGGUGCAAAAGUAAUUGCGGAUUUUGCCAUUAAAAGUAAUGGCAAAAAUAGCAAUUAUUUUUGUACCAGCCUAGUAUCUUUUCUCCUUCUACCAAACUUUGUCCCUGAGCCAUCUCA> hsa-mir-548i-2 MI0006422UAGAUGGCUCCGAAGUUUGCAUCCUAUUAGUUUGGUGCAAAAGUAAUUGCGGAUUUUGCCAUUAAAAGUAAUGGCAAAAAUAGCAAUUAUUUUUGUACCAGCCUAGUAUCUUUUCUCCUUCUAACAAAGUUCGUCCCUGAUCCAUCUCA> hsa-mir-548i-3 MI0006423CAGAUGGCUCCGAAGUUUACAUCCUAUUAGGUUUGUGCAAAAGUAAUUGCGGAUUUUGCCAUUAAAAGUAAUGGCAAAAAUAGCAAUUAUUUUUGUACCAGCCUAGUAUCUUUUCUCCUUCUACCAAACUUUGUCCCUGAGCCAUCUCA> hsa-mir-548i-4 MI0006424AGGUUGGUGCAAAAGUAAUUGCGGAUUUUGCCAUACUUUUAACGGCAAAAACCACAAAUAUUAUUGCACCAACCUAU> hsa-mir -548j MI0006345GGGCAGCCAGUGAAUAGUUAGCUGGUGCAAAAGUAAUUGCGGUCUUUGGUAUUACUUUCAGUGGCAAAAACUGCAUUACUUUGCACCAGCCUACUAGAACGCUGAGUUCAG> hsa-mir-548k MI0006354CUUUUCUCAU GCUUCAACCUAAUAUGAUGCAAAAUUGGCU> hsa-mir-548l MI0006361UAUUAGGUUGGUGCAAAAGUAUUUGCGGGUUUUGUCGUAGAAAGUAAUGGCAAAAACUGCAGUUACUUGUGCACCAACCAAAUGCU> hsa-mir-548m MI0006400AUAUUAGGUUGGUGCAAAGGUAUUUGUGGUUUUUGUCAUUAAAGUAAUGCAAAAGCCACAAAUACCUUUGCACCAACCUAAUAUUA> hsa-mir-548n MI0006399AGGUUGGUGCAAAAGUAAUUGUGGAUUUUGUCGUUAAAAAUAGCAAAACCCGCAAUUACUUUUGCACCAACCUAA> hsa-mir-548o MI0006402UGGUGAAAAUGUGUUGAUUGUAAUGGUUCCUAUUCUGAUCAAUAAACAUGGUUUGAGCCUAGUUACAAUGAUCUAAAAUUCACGGUCCAAAACUGCAGUUACUUUUGCACCAAC> hsa-mir-548o-2 MI0016746UGGUGCAAAAGUAAUUGCGGUUUUUGCCAUUAAAAGUAAUGCGGCCAAAACUGCAGUUACUUUUGCACCC> hsa-mir-548p MI0006420AUUAGGUUGGUAUAAAAUUAAUUGCAGUUUUUGUCAUUACUUUCAAUAGCAAAAACUGCAGUUACUUUUGCACCAAUGUAAUAC> hsa-mir- 548q MI0010637AUAUUAGGCUGGUGCAAAAGUAAUGGCGGUUUUUGCCAUUACUUUUCAUUUUUACCAUUAAAAGUAAUGGCAAAAAGCAUGAUUACUUUUUCACCAACCU> hsa-mir-548s MI0014141UUGCUGCAAAAAUAAUUGCAGUUUUUGCCAUUAUUUUUAAUAAUUAUAAUAAUGGCCAAAACUGCAGUUAUUUUUGCACCAA> hsa-mir-548t MI0014164AGGGUGGUGCAAAAGUGAUCGUGGUUUUUGCAAUUUUUUAAUGACAAAAACCACAAUUACUUUU GCACCAACCU> hsa-mir-548u MI0014168AUUAGGAUGGUGCAAAAGUAAUGUGGUUUUUUUCUUUACUUUUAAUGGCAAAGACUGCAAUUACUUUUGCGCCAACCUAAU> hsa-mir-548v MI0014174AAUACUAGGUUUGAGCAAAAGUAAUUGCGGUUUUGCCAUCAUGCCAAAAGCUACAGUUACUUUUGCACCAGCCUAAUAUU> hsa-mir-548w MI0014222GGUUGGUGCAAAAGUAACUGCGGUUUUUGCCUUUCAACAUAAUGGCAAAACCCACAAUUACUUUUGCACCAAUC> hsa-mir-548x MI0014244AGGUUAGUGCAAAAGUAAUUGCAGUUUUUGCGUUACUUUCAAUCGUAAAAACUGCAAUUACUUUCACACCAAUCU> hsa-mir-548x-2 MI0016833AUGCCAAAUAUUAGGUUGGCACAAAAGUAAUUGUGGCUUUUGCCAUUAAAAGUAAUGGUAAAAACUGCAAUUACUUUCGUGCCAACCUAAUAUUUGUGUG> hsa-mir-548y MI0016595GCCUAAACUAUUAGGUUGGUGCAAAAGUAAUCACUGUUUUUGCCAUUACUCUCAGUGGCAAAAACCGUGAUUACUUUUGCACCAACCUAGUAACACCUUCACUGUGGGGG> hsa-mir- 548z MI0016688AAGUAUUAAGUUGGUGCAAAAGUAAUUGAGAUUUUUGCUACUGAAAGUAAUGGCAAAAACCGCAAUUACUUUUGCACCAACCUAAUAGAUGCCAAUG> hsa-mir-549 MI0003679AGACAUGCAACUCAAGAAUAUAUUGAGAGCUCAUCCAUAGUUGUCACUGUCUCAAAUCAGUGACAACUAUGGAUGAGCUCUUAAUAUAUCCCAGGC> hsa-mir-550a-1 MI0003600UGAUGCUUUGCUGGCUGGUGCAGUGCCUGAGGGAGUAA
GAGCCCUGUUGUUGUAAGAUAGUGUCUUACUCCCUCAGGCACAUCUCCAACAAGUCUCU> hsa-mir-550a-2 MI0003601UGAUGCUUUGCUGGCUGGUGCAGUGCCUGAGGGAGUAAGAGCCCUGUUGUUGUCAGAUAGUGUCUUACUCCCUCAGGCACAUCUCCAGCGAGUCUCU> hsa-mir-550a-3 MI0003762GAUGCUUUGCUGGCUGGUGCAGUGCCUGAGGGAGUAAGAGUCCUGUUGUUGUAAGAUAGUGUCUUACUCCCUCAGGCACAUCUCCAACAAGUCUC> hsa-mir-550b-1 MI0016686AGAGACUUGUUGGAGAUGUGCCUGAGGGAGUAAGACACUAUCUUACAACAACAGGGCUCUUACUCCCUCAGGCACUGCACCAGCCAGCAAAGCAUCA> hsa-mir-550b-2 MI0016687AGAGACUCGCUGGAGAUGUGCCUGAGGGAGUAAGACACUAUCUGACAACAACAGGGCUCUUACUCCCUCAGGCACUGCACCAGCCAGCAAAGCAUCA> hsa-mir-551a MI0003556GGGGACUGCCGGGUGACCCUGGAAAUCCAGAGUGGGUGGGGCCAGUCUGACCGUUUCUAGGCGACCCACUCUUGGUUUCCAGGGUUGCCCUGGAAA> hsa-mir-551b MI0003575AGAUGUGCUCUCCUGGCCCAUGAAAUCAAGCGUGGGUGAGACCUGGUGCAGAACGGGAAGGCGACCCAUACUUGGUUUCAGAGGCUGUGAGAAUAA> hsa-mir-552 MI0003557AACCAUUCAAAUAUACCACAGUUUGUUUAACCUUUUGCCUGUUGGUUGAAGAUGCCUUUCAACAGGUGACUGGUUAGACAAACUGUGGUAUAUACA> hsa-mir-553 MI0003558CUUCAAUUUUAUUUUAAAACGGUGAGAUUUUGUUUUGUCUGAGAAAAUCUCGCUGUUUUAGACUGAGG> hsa-mir- 554 MI0003559ACCUGAGUAACCUUUGCUAGUCCUGACUCAGCCAGUACUGGUCUUAGACUGGUGAUGGGUCAGGGUUCAUAUUUUGGCAUCUCUCUCUGGGCAUCU> hsa-mir-555 MI0003561GGAGUGAACUCAGAUGUGGAGCACUACCUUUGUGAGCAGUGUGACCCAAGGCCUGUGGACAGGGUAAGCUGAACCUCUGAUAAAACUCUGAUCUAU> hsa-mir-556 MI0003562GAUAGUAAUAAGAAAGAUGAGCUCAUUGUAAUAUGAGCUUCAUUUAUACAUUUCAUAUUACCAUUAGCUCAUCUUUUUUAUUACUACCUUCAACA> hsa-mir-557 MI0003563AGAAUGGGCAAAUGAACAGUAAAUUUGGAGGCCUGGGGCCCUCCCUGCUGCUGGAGAAGUGUUUGCACGGGUGGGCCUUGUCUUUGAAAGGAGGUGGA> hsa-mir-558 MI0003564GUGUGUGUGUGUGUGUGUGGUUAUUUUGGUAUAGUAGCUCUAGACUCUAUUAUAGUUUCCUGAGCUGCUGUACCAAAAUACCACAAACGGGCUG> hsa-mir-559 MI0003565GCUCCAGUAACAUCUUAAAGUAAAUAUGCACCAAAAUUACUUUUGGUAAAUACAGUUUUGGUGCAUAUUUACUUUAGGAUGUUACUGGAGCUCCCA> hsa-mir-561 MI0003567CUUCAUCCACCAGUCCUCCAGGAACAUCAAGGAUCUUAAACUUUGCCAGAGCUACAAAGGCAAAGUUUAAGAUCCUUGAAGUUCCUGGGGGAACCAU> hsa-mir-562 MI0003568AGUGAAAUUGCUAGGUCAUAUGGUCAGUCUACUUUUAGAGUAAUUGUGAAACUGUUUUCAAAGUAGCUGUACCAUUUGCACUCCCUGUGGCAAU> hsa-mir-563 MI0003569AGCAAAGAAGUGUGUUGCCCUAGGAAAUGUGUGUUGCUGA UGUAAUUAGGUUGACAUACGUUUCCCUGGUAGCCA> hsa-mir-564 MI0003570CGGGCAGCGGGUGCCAGGCACGGUGUCAGCAGGCAACAUGGCCGAGAGGCCGGGGCCUCCGGGCGGCGCCGUGUCCGCGACCGCGUACCCUGAC> hsa-mir-566 MI0003572GCUAGGCGUGGUGGCGGGCGCCUGUGAUCCCAACUACUCAGGAGGCUGGGGCAGCAGAAUCGCUUGAACCCGGGAGGCGAAGGUUGCAGUGAGC> hsa-mir-567 MI0003573GGAUUCUUAUAGGACAGUAUGUUCUUCCAGGACAGAACAUUCUUUGCUAUUUUGUACUGGAAGAACAUGCAAAACUAAAAAAAAAAAAAGUUAUUGCU> hsa-mir-568 MI0003574GAUAUACACUAUAUUAUGUAUAAAUGUAUACACACUUCCUAUAUGUAUCCACAUAUAUAUAGUGUAUAUAUUAUACAUGUAUAGGUGUGUAUAUG> hsa-mir-569 MI0003576GGUAUUGUUAGAUUAAUUUUGUGGGACAUUAACAACAGCAUCAGAAGCAACAUCAGCUUUAGUUAAUGAAUCCUGGAAAGUUAAGUGACUUUAUUU> hsa-mir-570 MI0003577CUAGAUAAGUUAUUAGGUGGGUGCAAAGGUAAUUGCAGUUUUUCCCAUUAUUUUAAUUGCGAAAACAGCAAUUACCUUUGCACCAACCUGAUGGAGU> hsa-mir-571 MI0003578CCUCAGUAAGACCAAGCUCAGUGUGCCAUUUCCUUGUCUGUAGCCAUGUCUAUGGGCUCUUGAGUUGGCCAUCUGAGUGAGGGCCUGCUUAUUCUA > hsa-mir-572 MI0003579GUCGAGGCCGUGGCCCGGAAGUGGUCGGGGCCGCUGCGGGCGGAAGGGCGCCUGUGCUUCGUCCGCUCGGCGGUGGCCCAGCCAGGCCCGCGGGA> hsa-mir-573 MI0003580UU UAGCGGUUUCUCCCUGAAGUGAUGUGUAACUGAUCAGGAUCUACUCAUGUCGUCUUUGGUAAAGUUAUGUCGCUUGUCAGGGUGAGGAGAGUUUUUG> hsa-mir-574 MI0003581GGGACCUGCGUGGGUGCGGGCGUGUGAGUGUGUGUGUGUGAGUGUGUGUCGCUCCGGGUCCACGCUCAUGCACACACCCACACGCCCACACUCAGG> hsa-mir-575 MI0003582AAUUCAGCCCUGCCACUGGCUUAUGUCAUGACCUUGGGCUACUCAGGCUGUCUGCACAAUGAGCCAGUUGGACAGGAGCAGUGCCACUCAACUC> hsa-mir-576 MI0003583UACAAUCCAACGAGGAUUCUAAUUUCUCCACGUCUUUGGUAAUAAGGUUUGGCAAAGAUGUGGAAAAAUUGGAAUCCUCAUUCGAUUGGUUAUAACCA> hsa-mir-577 MI0003584UGGGGGAGUGAAGAGUAGAUAAAAUAUUGGUACCUGAUGAAUCUGAGGCCAGGUUUCAAUACUUUAUCUGCUCUUCAUUUCCCCAUAUCUACUUAC> hsa-mir-578 MI0003585AGAUAAAUCUAUAGACAAAAUACAAUCCCGGACAACAAGAAGCUCCUAUAGCUCCUGUAGCUUCUUGUGCUCUAGGAUUGUAUUUUGUUUAUAUAU> hsa-mir-579 MI0003586CAUAUUAGGUUAAUGCAAAAGUAAUCGCGGUUUGUGCCAGAUGACGAUUUGAAUUAAUAAAUUCAUUUGGUAUAAACCGCGAUUAUUUUUGCAUCAAC> hsa-mir-580 MI0003587AUAAAAUUUCCAAUUGGAACCUAAUGAUUCAUCAGACUCAGAUAUUUAAGUUAACAGUAUUUGAGAAUGAUGAAUCAUUAGGUUCCGGUCAGAAAUU > hsa-mir-581 MI0003588GUUAUGUGAAGGUAUUCUUGUGUUCUCUAGAUCAGUGCUUUUAGAAAAUUUG UGUGAUCUAAAGAACACAAAGAAUACCUACACAGAACCACCUGC> hsa-mir-582 MI0003589AUCUGUGCUCUUUGAUUACAGUUGUUCAACCAGUUACUAAUCUAACUAAUUGUAACUGGUUGAACAACUGAACCCAAAGGGUGCAAAGUAGAAACAUU> hsa-mir-583 MI0003590AACUCACACAUUAACCAAAGAGGAAGGUCCCAUUACUGCAGGGAUCUUAGCAGUACUGGGACCUACCUCUUUGGU> hsa-mir-584 MI0003591UAGGGUGACCAGCCAUUAUGGUUUGCCUGGGACUGAGGAAUUUGCUGGGAUAUGUCAGUUCCAGGCCAACCAGGCUGGUUGGUCUCCCUGAAGCAAC> hsa-mir-585 MI0003592UGGGGUGUCUGUGCUAUGGCAGCCCUAGCACACAGAUACGCCCAGAGAAAGCCUGAACGUUGGGCGUAUCUGUAUGCUAGGGCUGCUGUAACAA> hsa-mir-586 MI0003594AUGGGGUAAAACCAUUAUGCAUUGUAUUUUUAGGUCCCAAUACAUGUGGGCCCUAAAAAUACAAUGCAUAAUGGUUUUUCACUCUUUAUCUUCUUAU> hsa-mir-587 MI0003595CUCCUAUGCACCCUCUUUCCAUAGGUGAUGAGUCACAGGGCUCAGGGAAUGUGUCUGCACCUGUGACUCAUCACCAGUGGAAAGCCCAUCCCAUAU> hsa-mir-588 MI0003597AGCUUAGGUACCAAUUUGGCCACAAUGGGUUAGAACACUAUUCCAUUGUGUUCUUACCCACCAUGGCCAAAAUUGGGCCUAAG > hsa-mir-589 MI0003599UCCAGCCUGUGCCCAGCAGCCCCUGAGAACCACGUCUGCUCUGAGCUGGGUACUGCCUGUUCAGAACAAAUGCCGGUUCCCAGACGCUGCCAGCUGGCC> hsa-mir-590 MI0003602UAGCCAGUCAGAAAUGAGC UUAUUCAUAAAAGUGCAGUAUGGUGAAGUCAAUCUGUAAUUUUAUGUAUAAGCUAGUCUCUGAUUGAAACAUGCAGCA> hsa-mir-591 MI0003603UCUUAUCAAUGAGGUAGACCAUGGGUUCUCAUUGUAAUAGUGUAGAAUGUUGGUUAACUGUGGACUCCCUGGCUCUGUCUCAAAUCUACUGAUUC> hsa-mir-592 MI0003604UAUUAUGCCAUGACAUUGUGUCAAUAUGCGAUGAUGUGUUGUGAUGGCACAGCGUCAUCACGUGGUGACGCAACAUCAUGACGUAAGACGUCACAAC> hsa-mir-593 MI0003605CCCCCAGAAUCUGUCAGGCACCAGCCAGGCAUUGCUCAGCCCGUUUCCCUCUGGGGGAGCAAGGAGUGGUGCUGGGUUUGUCUCUGCUGGGGUUUCUCCU> hsa-mir-595 MI0003607ACGGAAGCCUGCACGCAUUUAACACCAGCACGCUCAAUGUAGUCUUGUAAGGAACAGGUUGAAGUGUGCCGUGGUGUGUCUGGAGGAAGCGCCUGU> hsa-mir-596 MI0003608AGCACGGCCUCUCCGAAGCCUGCCCGGCUCCUCGGGAACCUGCCUCCCGCAUGGCAGCUGCUGCCCUUCGGAGGCCG> hsa-mir-597 MI0003609UACUUACUCUACGUGUGUGUCACUCGAUGACCACUGUGAAGACAGUAAAAUGUACAGUGGUUCUCUUGUGGCUCAAGCGUAAUGUAGAGUACUGGUC> hsa-mir-598 MI0003610GCUUGAUGAUGCUGCUGAUGCUGGCGGUGAUCCCGAUGGUGUGAGCUGGAAAUGGGGUGCUACGUCAUCGUUGUCAUCGUCAUCAUCAUCAUCCGAG > hsa-mir-599 MI0003611AAAGACAUGCUGUCCAGUGUGUUUGAUAAGCUGACAUGGGACAGGGAUUCUUUCACUGUUGUGUCAGUUUAUCAAACCCAUACU UGGAUGAC> hsa-mir-600 MI0003613AAGUCACGUGCUGUGGCUCCAGCUUCAUAGGAAGGCUCUUGUCUGUCAGGCAGUGGAGUUACUUACAGACAAGAGCCUUGCUCAGGCCAGCCCUGCCC> hsa-mir-601 MI0003614UGCAUGAGUUCGUCUUGGUCUAGGAUUGUUGGAGGAGUCAGAAAAACUACCCCAGGGAUCCUGAAGUCCUUUGGGUGGA> hsa-mir-602 MI0003615UUCUCACCCCCGCCUGACACGGGCGACAGCUGCGGCCCGCUGUGUUCACUCGGGCCGAGUGCGUCUCCUGUCAGGCAAGGGAGAGCAGAGCCCCCCUG> hsa-mir-603 MI0003616GAUUGAUGCUGUUGGUUUGGUGCAAAAGUAAUUGCAGUGCUUCCCAUUUAAAAGUAAUGGCACACACUGCAAUUACUUUUGCUCCAACUUAAUACUU> hsa-mir-604 MI0003617AGAGCAUCGUGCUUGACCUUCCACGCUCUCGUGUCCACUAGCAGGCAGGUUUUCUGACACAGGCUGCGGAAUUCAGGACAGUGCAUCAUGGAGA> hsa-mir-605 MI0003618GCCCUAGCUUGGUUCUAAAUCCCAUGGUGCCUUCUCCUUGGGAAAAACAGAGAAGGCACUAUGAGAUUUAGAAUCAAGUUAGG> hsa-mir-606 MI0003619UGUAUCCUUGGUUUUUAGUAGUUUUACUAUGAUGAGGUGUGCCAUCCACCCCAUCAUAGUAAACUACUGAAAAUCAAAGAUACAAGUGCCUGACCA > hsa-mir-607 MI0003620UUGCCUAAAGUCACAGGUUAUAGAUCUGGAUUGGAACCCAGGGAGCCAGACUGCCUGGGUUCAAAUCCAGAUCUAUAACUUGUGACUUUGGG> hsa-mir-608 MI0003621GGGCCAAGGUGGGCCAGGGGUGGUUGGGACAGCUCCGU UCCAAGAGCUUCCAUCAAAGGCUGCCUCUUGGUGCAGCACAGGUAGA> hsa-mir-609 MI0003622UGCUCGGCUGUUCCUAGGGUGUUUCUCUCAUCUCUGGUCUAUAAUGGGUUAAAUAGUAGAGAUGAGGGCAACACCCUAGGAACAGCAGAGGAACC> hsa-mir-610 MI0003623UCUAUUUGUCUUAGGUGAGCUAAAUGUGUGCUGGGACACAUUUGAGCCAAAUGUCCCAGCACACAUUUAGCUCACAUAAGAAAAAUGGACUCUAGU> hsa-mir-611 MI0003624AAAAUGGUGAGAGCGUUGAGGGGAGUUCCAGACGGAGAUGCGAGGACCCCUCGGGGUCUGACCCACA> hsa-mir-612 MI0003625UCCCAUCUGGACCCUGCUGGGCAGGGCUUCUGAGCUCCUUAGCACUAGCAGGAGGGGCUCCAGGGGCCCUCCCUCCAUGGCAGCCAGGACAGGACUCUCA> hsa-mir-613 MI0003626GGUGAGUGCGUUUCCAAGUGUGAAGGGACCCUUCCUGUAGUGUCUUAUAUACAAUACAGUAGGAAUGUUCCUUCUUUGCCACUCAUACACCUUUA> hsa-mir-614 MI0003627UCUAAGAAACGCAGUGGUCUCUGAAGCCUGCAGGGGCAGGCCAGCCCUGCACUGAACGCCUGUUCUUGCCAGGUGGCAGAAGGUUGCUGC> hsa-mir-615 MI0003628CUCGGGAGGGGCGGGAGGGGGGUCCCCGGUGCUCGGAUCUCGAGGGUGCUUAUUGUUCGGUCCGAGCCUGGGUCUCCCUCUUCCCCCCAACCCCCC > hsa-mir-616 MI0003629UUAGGUAAUUCCUCCACUCAAAACCCUUCAGUGACUUCCAUGACAUGAAAUAGGAAGUCAUUGGAGGGUUUGAGCAGAGGAAUGACCUGUUUUAAAA> hsa-mir-617 MI0003631CAUCAUAAGGAGCCUAGAC UUCCCAUUUGAAGGUGGCCAUUUCCUACCACCUUCAAAUGGUAAGUCCAGGCUCCUUCUGAUUCAAUAAAUGAGGAGC> hsa-mir-618 MI0003632CUCUUGUUCACAGCCAAACUCUACUUGUCCUUCUGAGUGUAAUUACGUACAUGCAGUAGCUCAGGAGACAAGCAGGUUUACCCUGUGGAUGAGUCUGA> hsa-mir-619 MI0003633CGCCCACCUCAGCCUCCCAAAAUGCUGGGAUUACAGGCAUGAGCCACUGCGGUCGACCAUGACCUGGACAUGUUUGUGCCCAGUACUGUCAGUUUGCAG> hsa-mir-620 MI0003634AUAUAUAUCUAUAUCUAGCUCCGUAUAUAUAUAUAUAUAUAUAUAGAUAUCUCCAUAUAUAUGGAGAUAGAUAUAGAAAUAAAACAAGCAAAGAA> hsa-mir-621 MI0003635UAGAUUGAGGAAGGGGCUGAGUGGUAGGCGGUGCUGCUGUGCUCUGAUGAAGACCCAUGUGGCUAGCAACAGCGCUUACCUUUUGUCUCUGGGUCC> hsa-mir-622 MI0003636AGAGAAGCUGGACAAGUACUGGUCUCAGCAGAUUGAGGAGAGCACCACAGUGGUCAUCACACAGUCUGCUGAGGUUGGAGCUGCUGAGAUGACACU> hsa-mir-623 MI0003637GUACACAGUAGAAGCAUCCCUUGCAGGGGCUGUUGGGUUGCAUCCUAAGCUGUGCUGGAGCUUCCCGAUGUACUCUGUAGAUGUCUUUGCACCUUCUG> hsa-mir-624 MI0003638AAUGCUGUUUCAAGGUAGUACCAGUACCUUGUGUUCAGUGGAACCAAGGUAAACACAAGGUAUUGGUAUUACCUUGAGAUAGCAUUACACCUAAGUG > hsa-mir-625 MI0003639AGGGUAGAGGGAUGAGGGGGAAAGUUCUAUAGUCCUGUAAUUAGAUCUCAGGACUAUAGAACUUUCC CCCUCAUCCCUCUGCCCU> hsa-mir-626 MI0003640ACUGAUAUAUUUGUCUUAUUUGAGAGCUGAGGAGUAUUUUUAUGCAAUCUGAAUGAUCUCAGCUGUCUGAAAAUGUCUUCAAUUUUAAAGGCUU> hsa-mir-627 MI0003641UACUUAUUACUGGUAGUGAGUCUCUAAGAAAAGAGGAGGUGGUUGUUUUCCUCCUCUUUUCUUUGAGACUCACUACCAAUAAUAAGAAAUACUACUA> hsa-mir-628 MI0003642AUAGCUGUUGUGUCACUUCCUCAUGCUGACAUAUUUACUAGAGGGUAAAAUUAAUAACCUUCUAGUAAGAGUGGCAGUCGAAGGGAAGGGCUCAU> hsa-mir-629 MI0003643UCCCUUUCCCAGGGGAGGGGCUGGGUUUACGUUGGGAGAACUUUUACGGUGAACCAGGAGGUUCUCCCAACGUAAGCCCAGCCCCUCCCCUCUGCCU> hsa-mir-630 MI0003644AACUUAACAUCAUGCUACCUCUUUGUAUCAUAUUUUGUUAUUCUGGUCACAGAAUGACCUAGUAUUCUGUACCAGGGAAGGUAGUUCUUAACUAUAU> hsa-mir-631 MI0003645GUGGGGAGCCUGGUUAGACCUGGCCCAGACCUCAGCUACACAAGCUGAUGGACUGAGUCAGGGGCCACACUCUCC> hsa-mir-632 MI0003647CGCCUCCUACCGCAGUGCUUGACGGGAGGCGGAGCGGGGAACGAGGCCGUCGGCCAUUUUGUGUCUGCUUCCUGUGGGACGUGGUGGUAGCCGU > hsa-mir-633 MI0003648AACCUCUCUUAGCCUCUGUUUCUUUAUUGCGGUAGAUACUAUUAACCUAAAAUGAGAAGGCUAAUAGUAUCUACCACAAUAAAAUUGUUGUGAGGAUA> hsa-mir-634 MI0003649AAACCCACACCACUGCAUUU
UGGCCAUCGAGGGUUGGGGCUUGGUGUCAUGCCCCAAGAUAACCAGCACCCCAACUUUGGACAGCAUGGAUUAGUCU> hsa-mir-635 MI0003650CAGAGAGGAGCUGCCACUUGGGCACUGAAACAAUGUCCAUUAGGCUUUGUUAUGGAAACUUCUCCUGAUCAUUGUUUUGUGUCCAUUGAGCUUCCAAU> hsa-mir-636 MI0003651UGGCGGCCUGGGCGGGAGCGCGCGGGCGGGGCCGGCCCCGCUGCCUGGAAUUAACCCCGCUGUGCUUGCUCGUCCCGCCCGCAGCCCUAGGCGGCGUCG> hsa-mir-637 MI0003652UGGCUAAGGUGUUGGCUCGGGCUCCCCACUGCAGUUACCCUCCCCUCGGCGUUACUGAGCACUGGGGGCUUUCGGGCUCUGCGUCUGCACAGAUACUUC> hsa-mir-638 MI0003653GUGAGCGGGCGCGGCAGGGAUCGCGGGCGGGUGGCGGCCUAGGGCGCGGAGGGCGGACCGGGAAUGGCGCGCCGUGCGCCGCCGGCGUAACUGCGGCGCU> hsa-mir-639 MI0003654UGGCCGACGGGGCGCGCGCGGCCUGGAGGGGCGGGGCGGACGCAGAGCCGCGUUUAGUCUAUCGCUGCGGUUGCGAGCGCUGUAGGGAGCCUGUGCUG> hsa-mir-640 MI0003655GUGACCCUGGGCAAGUUCCUGAAGAUCAGACACAUCAGAUCCCUUAUCUGUAAAAUGGGCAUGAUCCAGGAACCUGCCUCUACGGUUGCCUUGGGG> hsa-mir-641 MI0003656UGGGUGAAAGGAAGGAAAGACAUAGGAUAGAGUCACCUCUGUCCUCUGUCCUCUACCUAUAGAGGUGACUGUCCUAUGUCUUUCCUUCCUCUUACCCCU > hsa-mir-642a MI0003657AUCUGAGUUGGGAGGGUCCCUCUCCAAAUGUGUCUUGGGGUGGGGGAUCAAGACACA UUUGGAGAGGGAACCUCCCAACUCGGCCUCUGCCAUCAUU> hsa-mir-642b MI0016685GAGUUGGGAGGUUCCCUCUCCAAAUGUGUCUUGAUCCCCCACCCCAAGACACAUUUGGAGAGGGACCCUCCCAACUC> hsa-mir-643 MI0003658ACCAAGUGAUAUUCAUUGUCUACCUGAGCUAGAAUACAAGUAGUUGGCGUCUUCAGAGACACUUGUAUGCUAGCUCAGGUAGAUAUUGAAUGAAAAA> hsa-mir-644a MI0003659UUUUUUUUUAGUAUUUUUCCAUCAGUGUUCAUAAGGAAUGUUGCUCUGUAGUUUUCUUAUAGUGUGGCUUUCUUAGAGCAAAGAUGGUUCCCUA> hsa-mir-644b MI0019134UGGGCUAAGGGAGAUGAUUGGGUAGAAAGUAUUAUUCUAUUCAUUUGCCUCCCAGCCUACA> hsa-mir-645 MI0003660CAGUUCCUAACAGGCCUCAGACCAGUACCGGUCUGUGGCCUGGGGGUUGAGGACCCCUGCUCUAGGCUGGUACUGCUGAUGCUUAAAAAGAGAG> hsa-mir-646 MI0003661GAUCAGGAGUCUGCCAGUGGAGUCAGCACACCUGCUUUUCACCUGUGAUCCCAGGAGAGGAAGCAGCUGCCUCUGAGGCCUCAGGCUCAGUGGC> hsa-mir-647 MI0003662AGGAAGUGUUGGCCUGUGGCUGCACUCACUUCCUUCAGCCCCAGGAAGCCUUGGUCGGGGGCAGGAGGGAGGGUCAGGCAGGGCUGGGGGCCUGAC > hsa-mir-648 MI0003663AUCACAGACACCUCCAAGUGUGCAGGGCACUGGUGGGGGCCGGGGCAGGCCCAGCGAAAGUGCAGGACCUGGCACUUAGUCGGAAGUGAGGGUG> hsa-mir-649 MI0003664GGCCUAGCCAAAUACUGUAUUUUGAUCGACAUUGGUUGAAAA GUAUUAGUAAACCUGUGUUGUUCAAGAGUCCACUGUGUUUUGCUG> hsa-mir-650 MI0003665CAGUGCUGGGGUCUCAGGAGGCAGCGCUCUCAGGACGUCACCACCAUGGCCUGGGCUCUGCUCCUCCUCACCCUCCUCACUCAGGGCACAGGUGAU> hsa-mir-651 MI0003666AAUCUAUCACUGCUUUUUAGGAUAAGCUUGACUUUUGUUCAAAUAAAAAUGCAAAAGGAAAGUGUAUCCUAAAAGGCAAUGACAGUUUAAUGUGUUU> hsa-mir-652 MI0003667ACGAAUGGCUAUGCACUGCACAACCCUAGGAGAGGGUGCCAUUCACAUAGACUAUAAUUGAAUGGCGCCACUAGGGUUGUGCAGUGCACAACCUACAC> hsa-mir-653 MI0003674UUCAUUCCUUCAGUGUUGAAACAAUCUCUACUGAACCAGCUUCAAACAAGUUCACUGGAGUUUGUUUCAAUAUUGCAAGAAUGAUAAGAUGGAAGC> hsa-mir-654 MI0003676GGGUAAGUGGAAAGAUGGUGGGCCGCAGAACAUGUGCUGAGUUCGUGCCAUAUGUCUGCUGACCAUCACCUUUAGAAGCCC> hsa-mir-655 MI0003677AACUAUGCAAGGAUAUUUGAGGAGAGGUUAUCCGUGUUAUGUUCGCUUCAUUCAUCAUGAAUAAUACAUGGUUAACCUCUUUUUGAAUAUCAGACUC> hsa-mir-656 MI0003678CUGAAAUAGGUUGCCUGUGAGGUGUUCACUUUCUAUAUGAUGAAUAUUAUACAGUCAACCUCUUUCCGAUAUCGAAUC > hsa-mir-657 MI0003681GUGUAGUAGAGCUAGGAGGAGAGGGUCCUGGAGAAGCGUGGACCGGUCCGGGUGGGUUCCGGCAGGUUCUCACCCUCUCUAGGCCCCAUUCUCCUCUG> hsa-mir-658 MI0003682GCUCGGUUGCCGUGGU UGCGGGCCCUGCCCGCCCGCCAGCUCGCUGACAGCACGACUCAGGGCGGAGGGAAGUAGGUCCGUUGGUCGGUCGGGAACGAGG> hsa-mir-659 MI0003683UACCGACCCUCGAUUUGGUUCAGGACCUUCCCUGAACCAAGGAAGAGUCACAGUCUCUUCCUUGGUUCAGGGAGGGUCCCCAACAAUGUCCUCAUGG> hsa-mir-660 MI0003684CUGCUCCUUCUCCCAUACCCAUUGCAUAUCGGAGUUGUGAAUUCUCAAAACACCUCCUGUGUGCAUGGAUUACAGGAGGGUGAGCCUUGUCAUCGUG> hsa-mir-661 MI0003669GGAGAGGCUGUGCUGUGGGGCAGGCGCAGGCCUGAGCCCUGGUUUCGGGCUGCCUGGGUCUCUGGCCUGCGCGUGACUUUGGGGUGGCU> hsa-mir-662 MI0003670GCUGUUGAGGCUGCGCAGCCAGGCCCUGACGGUGGGGUGGCUGCGGGCCUUCUGAAGGUCUCCCACGUUGUGGCCCAGCAGCGCAGUCACGUUGC> hsa-mir-663a MI0003672CCUUCCGGCGUCCCAGGCGGGGCGCCGCGGGACCGCCCUCGUGUCUGUGGCGGUGGGAUCCCGCGGCCGUGUUUUCCUGGUGGCCCGGCCAUG> hsa-mir-663b MI0006336GGUGCCGAGGGCCGUCCGGCAUCCUAGGCGGGUCGCUGCGGUACCUCCCUCCUGUCUGUGGCGGUGGGAUCCCGUGGCCGUGUUUUCCUGGUGGCCCGGCCGUGCCUGAGGUUUC> hsa-mir-664 MI0006442GAACAUUGAAACUGGCUAGGGAAAAUGAUUGGAUAGAAACUAUUAUUCUAUUCAUUUAUCCCCAGCCUACAAAAUGAAAAAA > hsa-mir-665 MI0005563UCUCCUCGAGGGGUCUCUGCCUCUACCCAGGACUCUUCAUGACCAGGAGGCUGAGGCCCCUCACAGGCG GC> hsa-mir-668 MI0003761GGUAAGUGCGCCUCGGGUGAGCAUGCACUUAAUGUGGGUGUAUGUCACUCGGCUCGGCCCACUACC> hsa-mir-670 MI0003933GUUUAGGGGUGGACCUGAUGUCCCUGAGUGUAUGUGGUGAACCUGAAUUUGCCUUGGGUUUCCUCAUAUUCAUUCAGGAGUGUCAGUUGCCCCUUCAC> hsa-mir-671 MI0003760GCAGGUGAACUGGCAGGCCAGGAAGAGGAGGAAGCCCUGGAGGGGCUGGAGGUGAUGGAUGUUUUCCUCCGGUUCUCAGGGCUCCACCUCUUUCGGGCCGUAGAGCCAGGGCUGGUGC> hsa-mir-675 MI0005416CCCAGGGUCUGGUGCGGAGAGGGCCCACAGUGGACUUGGUGACGCUGUAUGCCCUCACCGCUCAGCCCCUGGG> hsa-mir-676 MI0016436GCAUGACUCUUCAACCUCAGGACUUGCAGAAUUAAUGGAAUGCUGUCCUAAGGUUGUUGAGUUGUGC> hsa-mir-708 MI0005543AACUGCCCUCAAGGAGCUUACAAUCUAGCUGGGGGUAAAUGACUUGCACAUGAACACAACUAGACUGUGAGCUUCUAGAGGGCAGGGA> hsa-mir-711 MI0012488ACUGACUUUGAGUCUCUCCUCAGGGUGCUGCAGGCAAAGCUGGGGACCCAGGGAGAGACGUAAGUGAGGGGAGAUG > hsa-mir-718 MI0012489GGCCGCGGCGCGCAAGAUGGCGGCGGGCCCGGGCACCGCCCCUUCCGCCCCGCCGGGCGUCGCACGAGGC> hsa-mir-720 MI0006654CCGGAUCUCACACGGUGGUGUUAAUCUCGCUGGGCUCCAAAAUGUUGUGCCAG CGGGGCUAGGGCUAACAGCAGUCUUACUGAAGGUUUCCUGGAAACCACGCACAUGCUGUUGCCACUAACCUCAACCUUACUCGGUC> hsa-mir-758 MI0003757GCCUGGAUACAUGAGAUGGUUGACCAGAGAGCACACGCUUUAUUUGUGCCGUUUGUGACCUGGUCCACUAACCCUCAGUAUCUAAUGC> hsa-mir-759 MI0004065UAAUAAAUUAAAUGCCUAAACUGGCAGAGUGCAAACAAUUUUGACUCAGAUCUAAAUGUUUGCACUGGCUGUUUAAACAUUUAAUUUGUUA> hsa-mir-760 MI0005567GGCGCGUCGCCCCCCUCAGUCCACCAGAGCCCGGAUACCUCAGAAAUUCGGCUCUGGGUCUGUGGGGAGCGAAAUGCAAC> hsa-mir-761 MI0003941GGAGGAGCAGCAGGGUGAAACUGACACAGUUCUGGUGAGUUUCACUUUGCUGCUCCUCC> hsa-mir-762 MI0003892GGCCCGGCUCCGGGUCUCGGCCCGUACAGUCCGGCCGGCCAUGCUGGCGGGGCUGGGGCCGGGGCCGAGCCCGCGGCGGGGCC> hsa-mir-764 MI0003944AAUCUAGGAGGCAGGUGCUCACUUGUCCUCCUCCAUGCUUGGAAAAUGCAGGGAGGAGGCCAUAGUGGCAACUGUUACCAUGAUU> hsa-mir-765 MI0005116UUUAGGCGCUGAUGAAAGUGGAGUUCAGUAGACAGCCCUUUUCAAGCCCUACGAGAAACUGGGGUUUCUGGAGGAGAAGGAAGGUGAUGAAGGAUCUGUUCUCGUGAGCCUGAA > hsa-mir-766 MI0003836GCAUCCUCAGGACCUGGGCUUGGGUGGUAGGAGGAAUUGGUGCUGGUCUUUCAUUUGGAUUUGACUCCAGCCCCACAGCCUCACACCCCAGCCAAUUGUCAUAGGAGC> hsa-mir-767 MI0003763GCUUU UAUAUUGUAGGUUUUUGCUCAUGCACCAUGGUUGUCUGAGCAUGCAGCAUGCUUGUCUGCUCAUACCCCAUGGUUUCUGAGCAGGAACCUUCAUUGUCUACUGC> hsa-mir-769 MI0003834GCCUUGGUGCUGAUUCCUGGGCUCUGACCUGAGACCUCUGGGUUCUGAGCUGUGAUGUUGCUCUCGAGCUGGGAUCUCCGGGGUCUUGGUUCAGGGCCGGGGCCUCUGGGUUCCAAGC> hsa-mir-770 MI0005118AGGAGCCACCUUCCGAGCCUCCAGUACCACGUGUCAGGGCCACAUGAGCUGGGCCUCGUGGGCCUGAUGUGGUGCUGGGGCCUCAGGGGUCUGCUCUU> hsa-mir-802 MI0003906GUUCUGUUAUUUGCAGUCAGUAACAAAGAUUCAUCCUUGUGUCCAUCAUGCAACAAGGAGAAUCUUUGUCACUUAGUGUAAUUAAUAGCUGGAC> hsa-mir-873 MI0005564GUGUGCAUUUGCAGGAACUUGUGAGUCUCCUAUUGAAAAUGAACAGGAGACUGAUGAGUUCCCGGGAACACCCACAA> hsa-mir-874 MI0005532UUAGCCCUGCGGCCCCACGCACCAGGGUAAGAGAGACUCUCGCUUCCUGCCCUGGCCCGAGGGACCGACUGGCUGGGC> hsa-mir-875 MI0005541UUAGUGGUACUAUACCUCAGUUUUAUCAGGUGUUCUUAAAAUCACCUGGAAACACUGAGGUUGUGUCUCACUGAAC> hsa-mir-876 MI0005542UGAAGUGCUGUGGAUUUCUUUGUGAAUCACCAUAUCUAAGCUAAUGUGGUGGUGGUUUACAAAGUAAUUCAUAGUGCUUCA > hsa-mir-877 MI0005561GUAGAGGAGAUGGCGCAGGGGACACGGGCAAAGACUUGGGGGUUCCUGGGACCCUCAGACGUGUGUCCUCUUCUCCUCCUCCCAG> hsa-mir-885 MI0005560CCGCACUCUCUCCAUUACACUACCCUGCCUCUUCUCCAUGAGAGGCAGCGGGGUGUAGUGGAUAGAGCACGGGU> hsa-mir-887 MI0005562GUGCAGAUCCUUGGGAGCCCUGUUAGACUCUGGAUUUUACACUUGGAGUGAACGGGCGCCAUCCCGAGGCUUUGCACAG> hsa-mir-888 MI0005537GGCAGUGCUCUACUCAAAAAGCUGUCAGUCACUUAGAUUACAUGUGACUGACACCUCUUUGGGUGAAGGAAGGCUCA> hsa-mir-889 MI0005540GUGCUUAAAGAAUGGCUGUCCGUAGUAUGGUCUCUAUAUUUAUGAUGAUUAAUAUCGGACAACCAUUGUUUUAGUAUCC> hsa-mir-890 MI0005533GGAAGUGCCCUACUUGGAAAGGCAUCAGUUGCUUAGAUUACAUGUAACUAUUCCCUUUCUGAGUAGAGUAAGUCUUA> hsa-mir-891a MI0005524CCUUAAUCCUUGCAACGAACCUGAGCCACUGAUUCAGUAAAAUACUCAGUGGCACAUGUUUGUUGUGAGGGUCAAAAGA> hsa-mir-891b MI0005534CCUUAAUCCUUGCAACUUACCUGAGUCAUUGAUUCAGUAAAACAUUCAAUGGCACAUGUUUGUUGUUAGGGUCAAAAGA> hsa-mir-892a MI0005528GCAGUGCCUUACUCAGAAAGGUGCCAGUCACUUACACUACAUGUCACUGUGUCCUUUCUGCGUAGAGUAAGGCUC > hsa-mir-892b MI0005538UGCAAUGCCCUACUCAGAAAGGUGCCAUUUAUGUAGAUUUUAUGUCACUGGCUCCUUUCUGGGUAGAGCAAGGCUCA> hsa-mir-920 MI0005712GUAGUUGUUCUACAGAAGACCUGGAUGUGUAGGAGCUAAGACACUCCAGGAUGUGUAGGAG 05713ACUAGUGAGGGACAGAACCAGGAUUCAGACUCAGGUCCAUGGGCCUGGAUCACUGG> hsa-mir-922 MI0005714AUGGCGUUUUCCCUCUCCCUGUCCUGGACUGGGGUCAGACUGUGCCCCGAGGAGAAGCAGCAGAGAAUAGGACUACGUCAU> hsa-mir-924 MI0005716AAUAGAGUCUUGUGAUGUCUUGCUUAAGGGCCAUCCAACCUAGAGUCUACAAC> hsa-mir-933 MI0005755ACUUGGGUCAGUUCAGAGGUCCUCGGGGCGCGCGUCGAGUCAGCCGUGUGCGCAGGGAGACCUCUCCCACCCACAGU> hsa-mir-934 MI0005756AGAAAUAAGGCUUCUGUCUACUACUGGAGACACUGGUAGUAUAAAACCCAGAGUCUCCAGUAAUGGACGGGAGCCUUAUUUCU> hsa-mir-935 MI0005757GGCGGGGGCGCGGGCGGCAGUGGCGGGAGCGGCCCCUCGGCCAUCCUCCGUCUGCCCAGUUACCGCUUCCGCUACCGCCGCCGCUCCCGCU> hsa-mir-936 MI0005758UCAAGGCCACUGGGACAGUAGAGGGAGGAAUCGCAGAAAUCACUCCAGGAGCAACUGAGAGACCUUGCUUCUACUUUACCAGGUCCUGCUGGCCCAGA> hsa-mir-937 MI0005759AGCACUGCCCCCGGUGAGUCAGGGUGGGGCUGGCCCCCUGCUUCGUGCCCAUCCGCGCUCUGACUCUCUGCCCACCUGCAGGAGCU > hsa-mir-938 MI0005760GAAGGUGUACCAUGUGCCCUUAAAGGUGAACCCAGUGCACCUUCAUGAACCGUGGUACCUUUAAGAACUUGGUAUGCCUUC> hsa-mir-939 MI0005761UGUGGGCAGGGCCCUGGGGAGCUGGCUCUGGGGGUGGCCGGGGC r-940 MI0005762GUGAGGUGUGGGCCCGGCCCCAGGAGCGGGGCCUGGGCAGCCCCGUGUGUUGAGGAAGGAAGGCAGGGCCCCCGCUCCCCGGGCCUGACCCCAC> hsa-mir-941-1 MI0005763CACGGAAGAGGACACACCCGGCUGUGUGGACAUGUGCCCAGGGCCCGGGACAGCGCCACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGG> hsa-mir-941-2 MI0005764CACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGG> hsa-mir-941-3 MI0005765CACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCAUGG> hsa-mir-941-4 MI0005766CAUGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGG> hsa-mir-942 MI0005767AUUAGGAGAGUAUCUUCUCUGUUUUGGCCAUGUGUGUACUCACAGCCCCUCACACAUGGCCGAAACAGAGAAGUUACUUUCCUAAU> hsa- mir-943 MI0005768GGGACGUUCUGAGCUCGGGGUGGGGGACGUUUGCCGGUCUGCUGCUGGCGCCCUGACUGUUGCCGUCCUCCAGCCCCACUCAAAGGCAUCCC> hsa-mir-944 MI0005769GUUCCAGACACAUCUCAUCUGAUAUACAAUUUCUUAAUUGUA
AUUAUUGUACAUCGGAUGAGCUGUGUCUGGGAU> hsa-mir-1178 MI0006271GCGUUGGCUGGCAGAGGAAGGGAAGGGUCCAGGGUCAGCUGAGCAUGCCCUCAGGUUGCUCACUGUUCUUCCCUAGAAUGUCAGGUGAUGU> hsa-mir-1179 MI0006272GGCUGGAAAGGAAGAAGCAUUCUUUCAUUGGUUGGUGUGUAUUGCCUUGUCAACCAAUAAGAGGAUGCCAUUUAUCCUUUUCUGACUAGCU> hsa-mir-1180 MI0006273GCUGCUGGACCCACCCGGCCGGGAAUAGUGCUCCUGGUUGUUUCCGGCUCGCGUGGGUGUGUCGGCGGC> hsa-mir-1181 MI0006274UCCACUGCUGCCGCCGUCGCCGCCACCCGAGCCGGAGCGGGCUGGGCCGCCAAGGCAAGAUGGUGGACUACAGCGUGUGGG> hsa-mir-1182 MI0006275GGGACUUGUCACUGCCUGUCUCCUCCCUCUCCAGCAGCGACUGGAUUCUGGAGUCCAUCUAGAGGGUCUUGGGAGGGAUGUGACUGUUGGGAAGCCC> hsa-mir-1183 MI0006276AUUAUUCAAAUGCUCGGAGACACAGAACAUUAGAGAAGACAGGAGUUCACUGUAGGUGAUGGUGAGAGUGGGCAUGGAGCAGGAGUGCC> hsa-mir-1184- 1 MI0006277CUUGCAGAACGAGGUGAAGGAGGUGGUUCUGCUCAGCAGUCAACAGUGGCCACAUCUCCACCUGCAGCGACUUGAUGGCUUCCGUGUCCUUUUCGUGGG> hsa-mir-1184-2 MI0015971CUUGCAGAACGAGGUGAAGGAGGUGGUUCUGCUCAGCAGUCAACAGUGGCCACAUCUCCACCUGCAGCGACUUGAUGGCUUCCGUGUCCUUUUCGUGGG> hsa-mir-1184-3 MI0015972CUUGCAGAACGAGGUGAAGGAGGUGGUUCUGCUCAGCA GUCAACAGUGGCCACAUCUCCACCUGCAGCGACUUGAUGGCUUCCGUGUCCUUUUCGUGGG> hsa-mir-1185-1 MI0003844UUUGGUACUUGAAGAGAGGAUACCCUUUGUAUGUUCACUUGAUUAAUGGCGAAUAUACAGGGGGAGACUCUUAUUUGCGUAUCAAA> hsa-mir-1185-2 MI0003821UUUGGUACUUAAAGAGAGGAUACCCUUUGUAUGUUCACUUGAUUAAUGGCGAAUAUACAGGGGGAGACUCUCAUUUGCGUAUCAAA> hsa-mir-1193 MI0014205GUAGCUGAGGGGAUGGUAGACCGGUGACGUGCACUUCAUUUACGAUGUAGGUCACCCGUUUGACUAUCCACCAGCGCC> hsa-mir-1197 MI0006656ACUUCCUGGUAUUUGAAGAUGCGGUUGACCAUGGUGUGUACGCUUUAUUUGUGACGUAGGACACAUGGUCUACUUCUUCUCAAUAUCA> hsa-mir-1200 MI0006332UGCUACUUCUCCUGAGCCAUUCUGAGCCUCAAUCACUUGCCAGAGAGAUUGGUUCAGGAAUUUGUCAGGGAUAGCC> hsa-mir-1202 MI0006334CCUGCUGCAGAGGUGCCAGCUGCAGUGGGGGAGGCACUGCCAGGGCUGCCCACUCUGCUUAGCCAGCAGGUGCCAAGAACAGG> hsa- mir-1203 MI0006335UCCUCCCCGGAGCCAGGAUGCAGCUCAAGCCACAGCAGGGUGUUUAGCGCUCUUCAGUGGCUCCAGAUUGUGGCGCUGGUGCAGG> hsa-mir-1204 MI0006337ACCUCGUGGCCUGGUCUCCAUUAUUUGAGAUGAGUUACAUCUUGGAGGUG 0006339CAGUGUUCAUGUAGAUGUUUAAGCUCUUGCAGUAGGUUUUUGCAAGCUAGUGAACGCUG> hsa-mir-1207 MI0006340GCAGGGCUGGCAGGGAGGCUGGGAGGGGCUGGCUGGGUCUGGUAGUGGGCAUCAGCUGGCCCUCAUUUCUUAAGACAGCACUUCUGU> hsa-mir-1208 MI0006341CACCGGCAGAAUCACUGUUCAGACAGGCGGAGACGGGUCUUUCUCGCCCUCUGAUGAGUCACCACUGUGGUGG> hsa-mir-1224 MI0003764GUGAGGACUCGGGAGGUGGAGGGUGGUGCCGCCGGGGCCGGGCGCUGUUUCAGCUCGCUUCUCCCCCCACCUCCUCUCUCCUCAG> hsa-mir-1225 MI0006311GUGGGUACGGCCCAGUGGGGGGGAGAGGGACACGCCCUGGGCUCUGCCCAGGGUGCAGCCGGACUGACUGAGCCCCUGUGCCGCCCCCAG> hsa-mir-1226 MI0006313GUGAGGGCAUGCAGGCCUGGAUGGGGCAGCUGGGAUGGUCCAAAAGGGUGGCCUCACCAGCCCUGUGUUCCCUAG> hsa-mir-1227 MI0006316GUGGGGCCAGGCGGUGGUGGGCACUGCUGGGGUGGGCACAGCAGCCAUGCAGAGCGGGCAUUUGACCCCGUGCCACCCUUUUCCCCAG> hsa-mir-1228 MI0006318GUGGGCGGGGGCAGGUGUGUGGUGGGUGGUGGCCUGCGGUGAGCAGGGCCCUCACACCUGCCUCGCCCCCCAG > hsa-mir-1229 MI0006319GUGGGUAGGGUUUGGGGAGAGCGUGGGCUGGGGUUCAGGGACACCCUCUCACCACUGCCCUCCCACAG> hsa-mir-1231 MI0006321GUCAGUGUCUGGGCGGACAGCUGCAGGAAAGGGAAGACCAAGGCUUGCUGUCUGUCCAGUCUGCC CACAG> hsa-mir-1233-1 MI0006323GUGAGUGGGAGGCCAGGGCACGGCAGGGGGAGCUGCAGGGCUAUGGGAGGGGCCCCAGCGUCUGAGCCCUGUCCUCCCGCAG> hsa-mir-1233-2 MI0015973GUGAGUGGGAGGCCAGGGCACGGCAGGGGGAGCUGCAGGGCUAUGGGAGGGGCCCCAGCGUCUGAGCCCUGUCCUCCCGCAG> hsa-mir-1234 MI0006324GUGAGUGUGGGGUGGCUGGGGGGGGGGGGGGGGGGCCGGGGACGGCUUGGGCCUGCCUAGUCGGCCUGACCACCCACCCCACAG> hsa-mir-1236 MI0006326GUGAGUGACAGGGGAAAUGGGGAUGGACUGGAAGUGGGCAGCAUGGAGCUGACCUUCAUCAUGGCUUGGCCAACAUAAUGCCUCUUCCCCUUGUCUCUCCAG> hsa-mir-1237 MI0006327GUGGGAGGGCCCAGGCGCGGGCAGGGGUGGGGGUGGCAGAGCGCUGUCCCGGGGGCGGGGCCGAAGCGCGGCGACCGUAACUCCUUCUGCUCCGUCCCCCAG> hsa-mir-1238 MI0006328GUGAGUGGGAGCCCCAGUGUGUGGUUGGGGCCAUGGCGGGUGGGCAGCCCAGCCUCUGAGCCUUCCUCGUCUGUCUGCCCCAG> hsa- mir-1243 MI0006373CUAAAACUGGAUCAAUUAUAGGAGUGAAAUAAAGGUCCAUCUCCUGCCUAUUUAUUACUUUGCUUUGGUAAUAAAUCUAUUUUUAAAAGAACC> hsa-mir-1244-1 MI0006379AUCUUAUUCCGAGCAUUCCAGUAACUUUUUUGUGUAUGUACUUAGCUGUACUAUAAGUAGUUGGUUUGUAUGAGAUGGUUAAAAA> hsa-mir-1244-2 MI0015974AUCUUAUUCCGAGCAUUCCAGUAACUUUUUUGUGUAUGUACUUAGCUGUACUAUAAGUAGUUGGUUU GUAUGAGAUGGUUAAAAA> hsa-mir-1244-3 MI0015975AUCUUAUUCCGAGCAUUCCAGUAACUUUUUUGUGUAUGUACUUAGCUGUACUAUAAGUAGUUGGUUUGUAUGAGAUGGUUAAAAA> hsa-mir-1245a MI0006380AUUUAUGUAUAGGCCUUUAGAUCAUCUGAUGUUGAAUACUCUUUAAGUGAUCUAAAGGCCUACAUAUAAA> hsa-mir-1245b MI0017431UUUAUAUGUAGGCCUUUAGAUCACUUAAAGAGUAUUCAACAUCAGAUGAUCUAAAGGCCUAUACAUAAA> hsa-mir-1246 MI0006381UGUAUCCUUGAAUGGAUUUUUGGAGCAGGAGUGGACACCUGACCCAAAGGAAAUCAAUCCAUAGGCUAGCAAU> hsa-mir-1247 MI0006382CCGCUUGCCUCGCCCAGCGCAGCCCCGGCCGCUGGGCGCACCCGUCCCGUUCGUCCCCGGACGUUGCUCUCUACCCCGGGAACGUCGAGACUGGAGCGCCCGAACUGAGCCACCUUCGCGGACCCCGAGAGCGGCG> hsa-mir-1248 MI0006383UUUACCUUCUUGUAUAAGCACUGUGCUAAAAUUGCAGACACUAGGACCAUGUCUUGGUUUUUGCAAUAAUGCUAGCAGAGUACACACAAGAAGAAAAGUAACAGCA> hsa-mir- 1249 MI0006384GGGAGGAGGGAGGAGAUGGGCCAAGUUCCCUCUGGCUGGAACGCCCUUCCCCCCCUUCUUCACCUG> hsa-mir-1250 MI0006385CUGUCCCGCUGGCCUGGCAGGUGACGGUGCUGGAUGUGGCCUUUUGCCUUUCUAAAGGCCACAUUC GCCAGUGUAGACAUGGC> hsa-mir-1252 MI0006434AGAAAGAAGGAAAUUGAAUUCAUUUAGAAAAGAGAAUUCCAAAUGAGCUUAAUUUCCUUUUUUCU> hsa-mir-1253 MI0006387AGCAGCAAGAGAUAGAAUCCAAAAGAGAAGAAGAUCAGCCUGCAGAUGUGGACUGCUAAAUGCAGGCUGAUCUUCUCCCCUUUGGGAUUCUCUUAUGAGAAGCCA> hsa-mir-1254-1 MI0006388GGUGGGAGGAUUGCUUGAGCCUGGAAGCUGGAGCCUGCAGUGAACUAUCAUUGUGCCACUGUACUCCAGCCUAGGCAACAAAAUGAAAUCCUGUCUA> hsa-mir-1254-2 MI0016747CUGAGCCUGGAAGCUGGAGCCUGCAGUGAGCUAUGAUCAUGUCCCUGUACUCUAGCCUGGGCA> hsa-mir-1255a MI0006389AUUGGAAAUCCUUUGAGUUGCUUCUCAAGGAUGAGCAAAGAAAGUAGAUUUUUUAGAUUCUAAAGAAACUAUCUUCUUUGCUCAUCCUUGAGAAGCAACUCCUUAUCCAUUAA> hsa-mir-1255b-1 MI0006435UACGGAUGAGCAAAGAAAGUGGUUUCUUAAAAUGGAAUCUACUCUUUGUGAAGAUGCUGUGAA> hsa-mir-1255b-2 MI0006436UCUUACGGAUGAGCAAAGAAAGUGGUUUGCGCCUCAAGAAACCACUUUCUUUGCUCAUCCAUAAGGA> hsa-mir-1256 MI0006390AGUCAGCCUGUUGAAGCUUUGAAGCUUUGAUGCCAGGCAUUGACUUCUCACUAGCUGUGAAAGUCCUAGCUAAAGAGAAGUCAAUGCAUGACAUCUUGUUUCAAUAGAUGGCUGUUUCA> hsa-mir-1257 MI0006391GCCCUGGGCUUGUGCUUGGGGAGUGAAUGAUGGGUUCUGACCCCCAUGCACCCCUGUGGGCCCCUGGCAUCAC UGGCCCCAUCCUUCACCCCUGCCAACCACGCUUGCCCUGUGCCU> hsa-mir-1258 MI0006392CUGUGGCUUCCACGACCUAAUCCUAACUCCUGCGAGUCCCUGGAGUUAGGAUUAGGUCGUGGAAGCCACAGGA> hsa-mir-1260a MI0006394ACCUUUCCAGCUCAUCCCACCUCUGCCACCAAAACACUCAUCGCGGGGUCAGAGGGAGUGCCAAAAAAGGUAA> hsa-mir-1260b MI0014197UCUCCGUUUAUCCCACCACUGCCACCAUUAUUGCUACUGUUCAGCAGGUGCUGCUGGUGGUGAUGGUGAUAGUCUGGUGGGGGCGGUGG> hsa-mir-1261 MI0006396UGCUAUGGAUAAGGCUUUGGCUUAUGGGGAUAUUGUGGUUGAUCUGUUCUAUCCAGAUGACUGAAACUUUCUCCAUAGCAGC> hsa-mir-1262 MI0006397AUCUACAAUGGUGAUGGGUGAAUUUGUAGAAGGAUGAAAGUCAAAGAAUCCUUCUGGGAACUAAUUUUUGGCCUUCAACAAGAAUUGUGAUAU> hsa-mir-1263 MI0006398CUACCCCAAAAUAUGGUACCCUGGCAUACUGAGUAUUUUAAUACUGGCAUACUCAGUAUGCCAUGUUGCCAUAUUUUGGGGUAGCA> hsa-mir-1264 MI0003758AGGUCCUCAAUAAGUAUUUGUUGAAAGAAUAAAUAAACCAACAAGUCUUAUUUGAGCACCUGUUAUGUG > hsa-mir-1265 MI0006401AUGGUUUGGGACUCAGGAUGUGGUCAAGUGUUGUUAAGGCAUGUUCAGGAACAAUACUUGACCACAUUUUGAAUUCCAAACCAUAU> hsa-mir-1266 MI0006403ACAGGUAGUGUCCCUCAGCGCUGUAGAACAGGGCUG 7 MI0006404CUCCCAAAUCUCCUGUUGAAGUGUAAUCCCCACCUCCAGCAUUGGGGAUUACAUUUCAACAUGAGAUUUGGAUGAGGA> hsa-mir-1268a MI0006405UAGCCGGGCGUGGUGGUGGGGGCCUGUGGUCCCAGCUACUUUGGAGGCUGAG> hsa-mir-1268b MI0016748ACCCGGGCGUGGUGGUGGGGGUGGGUGCCUGUAAUUCCAGCUAGUUGGGA> hsa-mir-1269a MI0006406UGGAUUGCCUAGACCAGGGAAGCCAGUUGGCAUGGCUCAGUCCAAGUCUGACCACCUGAGGAAUGCCUGGACUGAGCCGUGCUACUGGCUUCCCUGGUCUCCAGC> hsa-mir-1269b MI0016888UGAGGUUUCUGGACUGAGCCAUGCUACUGGCUUCUCUGGUUCUCCAGCUUACAGAUGGCUUAUCAUGGGACCUCU> hsa-mir-1270-1 MI0006407CACAGAGUUAUACUGGAGAUAUGGAAGAGCUGUGUUGGGUAUAAGUAACAGGCUUUUCUUUAUCUUCUAUGUGGCUCUUUGCA> hsa-mir-1270-2 MI0015976CACAGAGUUAUACUGGAGAUAUGGAAGAGCUGUGUUGGGUAUAAGUAACAGGCUUUUCUUUAUCUUCUAUGUGGCUCUUUGCA> hsa -mir-1271 MI0003814CACCCAGAUCAGUGCUUGGCACCUAGCAAGCACUCAGUAAAUAUUUGUUGAGUGCCUGCUAUGUGCCAGGCAUUGUGCUGAGGGCU> hsa-mir-1272 MI0006408CCAGAUCAGAUCUGGGUGCGAUGAUGAUGGCAGCAAAUUCUGAAAACGUGCUCAGUGUCUUUAUAACAGGAAAGCCGUAAACUUAGAAAUGUAGGCUGCAGCUCGUGUGCUCUGUGGUCUGGGCUGGUA> hsa-mir-1273a MI0006409UGAGGCAGGAGAAUUGCUUGAACCCGGGUGGU GGAGGUUGCAGUGAGCCAAGAUUGCGCCACUGCACUCCAGCCUGGGCGACAAAGCAAGACUCUUUCUUGGA> hsa-mir-1273c MI0014171UGCAGCCUGGGCGACAAAACGAGACCCUGUCUUUUUUUUUUUCUGAGACAGAGUCUCGUUCUGUUGCCCAAGCUGGA> hsa-mir-1273d MI0014254GAAUCGCUUGAACCCAUGAGGUUGAGGCUGCAGUGAGCCAAGAUCGUGCCACUGCACUUCAGCCUGGGUGACAAGAGCGAAACUUC> hsa-mir-1273e MI0016059UGAGGCAGGAGAAUUGCUUGAACCCAGGAAGUGGAGGCUGCAGUGAGCCGAGAUCGAGCCACUGUACUCCAGCCUGGGUGACACAGCGAGACUCCAGUCUCA> hsa-mir-1273f MI0018002AGGUGGGAGGAUUGCUUGAGCCUGGGAGAUGGAGGUUGCAGUGAGCUGAGAUCACGCAACUGCACCCCCAGCCUGGGCCAUAGAGUCAGUCCUUGUCUC> hsa-mir-1273g MI0018003GAGGUGGGAGGAUUGCUUGAGUCAGGGUGGUUGAGGCUGCAGUAAGUUGUGAUCAUACCACUGCACUCCAGCCUGAGUGACAGAGCAAGACCUUGUCUCA> hsa-mir-1275 MI0006415CCUCUGUGAGAAAGGGUGUGGGGGAGAGGCUGUCUUGUGUCUGUAAGUAUGCCAAACUUAUUUUCCCCAAGGCAGAGGGA> hsa-mir-1276 MI0006416CCCCAGCUAGGUAAAGAGCCCUGUGGAGACACCUGGAUUCAGAGAACAUGUCUCCACUGAGCACUUGGGCCUUGAUGGCGGCU > hsa-mir-1277 MI0006419ACCUCCCAAAUAUAUAUAUAUAUGUACGUAUGUGUAUAUAAAUGUAUACGUAGAUAUAUGUAUUUUGGUGGGUUU> hsa-mir-1278 MI0006425AUUUGCUCAUAG AUGAUAUGCAUAGUACUCCCAGAACUCAUUAAGUUGGUAGUACUGUGCAUAUCAUCUAUGAGCGAAUAG> hsa-mir-1279 MI0006426AUAUUCACAAAAAUUCAUAUUGCUUCUUUCUAAUGCCAAGAAAGAAGAGUAUAAGAACUUCC> hsa-mir-1280 MI0006437UCUGUCCCACCGCUGCCACCCUCCCCUCUGCCUCAGUGUGCCAGGCAUCAGCACUCACUCACAGAGGCAGGCUGGAUGGCGGGUGGGACAACAG> hsa-mir-1281 MI0006428AGGGGGCACCGGGAGGAGGUGAGUGUCUCUUGUCGCCUCCUCCUCUCCCCCCUU> hsa-mir-1282 MI0006429CCUUCUUCUCGUUUGCCUUUUUCUGCUUCUGCUGCAUGAUCUCCGAGUCCCUGGGGGUAGAGAUGAUGGGGCACUGGGAGGUACCAGAGGGCAAAAAGGAC> hsa-mir-1283-1 MI0003832CUCAAGCUAUGAGUCUACAAAGGAAAGCGCUUUCUGUUGUCAGAAAGAAGAGAAAGCGCUUCCCUUUUGAGGGUUACGGUUUGAGAA> hsa-mir-1283-2 MI0006430CUCAAGCUGUGAGUCUACAAAGGAAAGCGCUUUCUGUUGUCUGAAAGAAAAGAAAUCGCUUCCCUUUGGAGUGUUACGGUUUGAGAA> hsa- mir-1284 MI0006431AUUUUGAUAUAUAAGCCAGUUUAAUGUUUCUAUACAGACCCUGGCUUUUCUUAAAUUUAUAUAUUGGAAAGCCCAUGUUUGUAUUGGAAACUGCUGGUUUCUUUCAUACUGAAAAUCU> hsa-mir-1285-1 MI0006346UGUAGA
I0006347UUUGGGAGGCCGAGGCUGGUGCAUCACUUGAGCCCAGCAAUUUGAGACCAAUCUGGGCAACAAAGUGAGACCUCCGUCUCUACAAAGA> hsa-mir-1286 MI0006348UGUCCUCUGGGGACUCAGCUUGCUCUGGCUGCUGGAUUGAAUUAGCUGCAGGACCAAGAUGAGCCCUUGGUGGAGACA> hsa-mir-1287 MI0006349GUUGUGCUGUCCAGGUGCUGGAUCAGUGGUUCGAGUCUGAGCCUUUAAAAGCCACUCUAGCCACAGAUGCAGUGAUUGGAGCCAUGACAA> hsa-mir-1288 MI0006432GAGGGUGUUGAUCAGCAGAUCAGGACUGUAACUCACCAUAGUGGUGGACUGCCCUGAUCUGGAGACCACUGCCUU> hsa-mir-1289-1 MI0006350UUCUCAAUUUUUAGUAGGAAUUAAAAACAAAACUGGUAAAUGCAGACUCUUGGUUUCCACCCCCAGAGAAUCCCUAAACCGGGGGUGGAGUCCAGGAAUCUGCAUUUUAGAAAGUACCCAGGGUGAUUCUGAUAAUUGGGAACA> hsa-mir-1289-2 MI0006351CCACGGUCCUAGUUAAAAAGGCACAUUCCUAGACCCUGCCUCAGAACUACUGAACAGAGUCACUGGGUGUGGAGUCCAGGAAUCUGCAUUUUUACCCCUAUCGCCCCCGCC> hsa-mir-1290 MI0006352GAGCGUCACGUUGACACUCAAAAAGUUUCAGAUUUUGGAACAUUUCGGAUUUUGGAUUUUUGGAUCAGGGAUGCUCAA> hsa- mir-1291 MI0006353GGUAGAAUUCCAGUGGCCCUGACUGAAGACCAGCAGUUGUACUGUGGCUGUUGGUUUCAAGCAGAGGCCUAAAGGACUGUCUUCCUG> hsa-mir-1292 MI0006433CCUGGGAACGGGUUCCGGCAGACGCUGGUUGCGUUGACGCUCGCGCCGG CUCCCGUUCCAGG> hsa-mir-1293 MI0006355AGGUUGUUCUGGGUGGUCUGGAGAUUUGUGCAGCUUGUACCUGCACAAAUCUCCGGACCACUUAGUCUUUA> hsa-mir-1294 MI0006356CACCUAAUGUGUGCCAAGAUCUGUUCAUUUAUGAUCUCACCGAGUCCUGUGAGGUUGGCAUUGUUGUCUGGCAUUGUCUGAUAUACAACAGUGCCAACCUCACAGGACUCAGUGAGGUGAAACUGAGGAUUAGGAAGGUGUA> hsa-mir-1295a MI0006357AGGACAUUUUGCCCAGAUCCGUGGCCUAUUCAGAAAUGUGGCCUGUGAUUAGGCCGCAGAUCUGGGUGAAAUGUCCUCC> hsa-mir-1295b MI0019146CACCCAGAUCUGCGGCCUAAUCACAGGCCACAUUUCUGAAUAGGCCACGGAUCUGGGCAA> hsa-mir-1296 MI0003780ACCUACCUAACUGGGUUAGGGCCCUGGCUCCAUCUCCUUUAGGAAAACCUUCUGUGGGGAGUGGGGCUUCGACCCUAACCCAGGUGGGCUGU> hsa-mir-1297 MI0006358UGUUUAUCUCUAGGGUUGAUCUAUUAGAAUUACUUAUCUGAGCCAAAGUAAUUCAAGUAAUUCAGGUGUAGUGAAAC> hsa-mir-1298 MI0003938AGACGAGGAGUUAAGAGUUCAUUCGGCUGUCCAGAUGUAUCCAAGUACCCUGUGUUAUUUGGCAAUAAAUACAUCUGGGCAACUGACUGAACUUUUCACUUUUCAUGACUCA > hsa-mir-1299 MI0006359CCUCAUGGCAGUGUUCUGGAAUCCUACGUGAGGGACAAUCAUUCAGACCCACGUAGCAGUGUUCUGGAAUUCUGUGUGAGGGA> hsa-mir-1301 MI0003815GGAUUGUGGGGGGUCGCUAGGCGCAGCACUGUGCUGG AGUGACUUCACACAGUCCUC> hsa-mir-1302-1 MI0006362CAGAAAGCCCAGUUAAAUUUGAAUUUCAAGUAAACAAUGAAUAAUUGUGUAUGUAAGAAUAUCCCAUACAAUAUUUGGGACAUACUUAUGCUAAAAAUUAUUCCUUGCUUAUCUGAAAUUCAAAUGUAACUAGGAUUCCUGUA> hsa-mir-1302-10 MI0015979GGAUGCCCAGCUAGUUUGAAUUUUAGAUAAACAACGAAUAAUUUCGUAGCAUAAAUAUGUCCCAAGCUUAGUUUGGGACAUACUUAUGCUAAAAAACAUUAUUGGUUGUUUAUCUGAGAUUCAGAAUUAAGCAUUUUA> hsa-mir-1302-11 MI0015980GGAUGCCCAGCUAGUUUGAAUUUUAGAUAAACAACGAAUAAUUUCGUAGCAUAAAUAUGUCCCAAGCUUAGUUUGGGACAUACUUAUGCUAAAAAACAUUAUUGGUUGUUUAUCUGAGAUUCAGAAUUAAGCAUUUUA> hsa-mir-1302-2 MI0006363GGAUGCCCAGCUAGUUUGAAUUUUAGAUAAACAACGAAUAAUUUCGUAGCAUAAAUAUGUCCCAAGCUUAGUUUGGGACAUACUUAUGCUAAAAAACAUUAUUGGUUGUUUAUCUGAGAUUCAGAAUUAAGCAUUUUA> hsa-mir-1302-3 MI0006364GGAUGCCCAGCUAGUUUGAAUUUUAGAUAAACAACGAAUAAUUUCGUAGCAUAAAUAUUUCCCAAGCUUAGUUUGGGACAUACUUAUGCUAAAAAACAUUAUUGGUUGUUUAUCUGAGAUUCAAAAUUAAGCAUUUUA> hsa-mir -1302-4 MI0006365AAUGCAGAAGCACAGCUAAAAUUUGAAUUUCAGAUAAACAAAUUUUUCUUAGAAUAAGUAUGUCCAUGCAACAUUUGGACAUACUUAUGCUAAAAUAUAUAUUGUGUUCAUCUGAAAUUCAAAUUCAAC UGGACAUCCUGUAUUUU> hsa-mir-1302-5 MI0006366UGCCCGGCCUCCCAUUAAAUUGGUUUUUCAGACAAAUCACAAAUUUGUUUAGGUAUAAGUAUAUCCCAUGUAAUCUUUGGGACAUACUUAUGCUAAAAUAAUUGUUCCUUGUUGAUUGGAAAUUUUAAUUUUAAUUAGGUGUCCUGUAUU> hsa-mir-1302-6 MI0006367AACAAAUAAUUUGGUAAUAUAUGUAUGGCCCACACAAUAUUUAGGACAACAAUAUUUGGGACAUACUUAUGCUAAAAAAGUAUUUGUUGA> hsa-mir-1302-7 MI0006368ACAACAUGUUUUUAGGACAUGUAUGUCUGGUGCAAUAAUUGGGACAUACUUAUGCUAAAAAAAUUAGUGUUC> hsa-mir-1302-8 MI0006369CCCAUUUAAACUUGAAUUUCAUAUAAACACCGUAAUUUUCAGCAUUAGUGUAUCACAUGCAGUAUUUGGGACAUACUUAUGCUAAAAAAUUAGGUGGUGUUGAUCUGAAAUUCCAGUGUAGAUGGGCA> hsa-mir-1302-9 MI0015978GGAUGCCCAGCUAGUUUGAAUUUUAGAUAAACAACGAAUAAUUUCGUAGCAUAAAUAUGUCCCAAGCUUAGUUUGGGACAUACUUAUGCUAAAAAACAUUAUUGGUUGUUUAUCUGAGAUUCAGAAUUAAGCAUUUUA> hsa-mir -1303 MI0006370GGCUGGGCAACAUAGCGAGACCUCAACUCUACAAUUUUUUUUUUUUAAAUUUUAGAGACGGGGUCUUGCUCUGUUGCCAGGCUUU> hsa-mir-1304 MI0006371AAACACUUGACCCAGCGGUUUGAGGCUA UUUAUUGUAAAGAUACUUUUCAACUCUAAUGGGAGAGACAGCAGGAUUCUCC> hsa-mir-1306 MI0006443GUGAGCAGUCUCCACCACCUCCCCUGCAAACGUCCAGUGGUGCAGAGGUAAUGGACGUUGGCUCUGGUGGUGAUGGACAGUCCGA> hsa-mir-1307 MI0006444CAUCAAGACCCAGCUGAGUCACUGUCACUGCCUACCAAUCUCGACCGGACCUCGACCGGCUCGUCUGUGUUGCCAAUCGACUCGGCGUGGCGUCGGUCGUGGUAGAUAGGCGGUCAUGCAUACGAAUUUUCAGCUCUUGUUCUGGUGAC> hsa-mir-1321 MI0006652ACAUUAUGAAGCAAGUAUUAUUAUCCCUGUUUUACAAAUAAGGAAAUAAACUCAGGGAGGUGAAUGUGAUCAAAGAUAG> hsa-mir-1322 MI0006653AGUAUCAUGAAUUAGAAACCUACUUAUUACAUAGUUUACAUAAGAAGCGUGAUGAUGCUGCUGAUGCUGUA> hsa-mir-1323 MI0003786ACUGAGGUCCUCAAAACUGAGGGGCAUUUUCUGUGGUUUGAAAGGAAAGUGCACCCAGUUUUGGGGAUGUCAA> hsa-mir-1324 MI0006657CCUGAAGAGGUGCAUGAAGCCUGGUCCUGCCCUCACUGGGAACCCCCUUCCCUCUGGGUACCAGACAGAAUUCUAUGCACUUUCCUGGAGGCUCCA> hsa-mir-1343 MI0017320GCUGGCGUCGGUGCUGGGGAGCGGCCCCCGGGUGGGCCUCUGCUCUGGCCCCUCCUGGGGCCCGCACUCUCGCUCUGGGCCCGC > hsa-mir-1468 MI0003782GGUGGGUGGUUUCUCCGUUUGCCUGUUUCGCUGAUGUGCAUUCAACUCAUUCUCAGCAAAAUAAGCAAAUGGAAAAUUCGUCCAUC> hsa-mir-1469 MI0007074CUCGGCGCGGGGCGCGGG CUCCGGGUUGGGGCGAGCCAACGCCGGGG> hsa-mir-1470 MI0007075GCCCUCCGCCCGUGCACCCCGGGGCAGGAGACCCCGCGGGACGCGCCGAGGUAGGGGGGAC> hsa-mir-1471 MI0007076GCCCGCGUGUGGAGCCAGGUGUAGAGGCGGAGCACAGCUGGCUCUAAUUUGAGGGGC> hsa-mir-1537 MI0007258ACAGCUGUAAUUAGUCAGUUUUCUGUCCUGUCCACACAGAAAACCGUCUAGUUACAGUUGU> hsa-mir-1538 MI0007259GGGAACAGCAGCAACAUGGGCCUCGCUUCCUGCCGGCGCGGCCCGGGCUGCUGCUGUUCCU> hsa-mir-1539 MI0007260GGCUCUGCGGCCUGCAGGUAGCGCGAAAGUCCUGCGCGUCCCAGAUGCCC> hsa-mir-1587 MI0016905UUUGGGCUGGGCUGGGUUGGGCAGUUCUUCUGCUGGACUCACCUGUGACCAGC> hsa-mir-1825 MI0008193AGAGACUGGGGUGCUGGGCUCCCCUAGACUAGGACUCCAGUGCCCUCCUCUCC > hsa-mir-1827 MI0008195UCAGCAGCACAGCCUUCAGCCUAAAGCAAUGAGAAGCCUCUGAAAGGCUGAGGCAGUAGAUUGAAU> hsa-mir-1908 MI0008329CGGGAAUGCCGCGGCGGGGACGGCGAUUGGUCCGUAUGUGUGGUGCCACCGGCCGCCGGCUCCGCCCCGGCCCCCGCCCC> hsa-mir-1909 MI0008330CAUCCAGGACAAUGGUGAGUGCCGGUGCCUGCCCUGGGGCCGUCCCUGCGCAGGGGCCGGGUGCUCACCGCAUCUGCCCC> hsa-mir-1910 MI0008331UGUCCCUUCAGCCAGUCCUGUGCCUGCCGCCUUUGUGCUGUCCUUGGAGGGAGGCAGAAGCAGGAUGACAAUGAGGGCAA> hsa-mir-1911 MI 0008332UCGGCAUCUGCUGAGUACCGCCAUGUCUGUUGGGCAUCCACAGUCUCCCACCAGGCAUUGUGGUCUCCGCUGACGCUUUG> hsa-mir-1912 MI0008333CUCUAGGAUGUGCUCAUUGCAUGGGCUGUGUAUAGUAUUAUUCAAUACCCAGAGCAUGCAGUGUGAACAUAAUAGAGAUU> hsa-mir-1913 MI0008334ACCUCUACCUCCCGGCAGAGGAGGCUGCAGAGGCUGGCUUUCCAAAACUCUGCCCCCUCCGCUGCUGCCAAGUGGCUGGU> hsa-mir-1914 MI0008335CGUGUGAGCCCGCCCUGUGCCCGGCCCACUUCUGCUUCCUCUUAGCGCAGGAGGGGUCCCGCACUGGGAGGGGCCCUCAC> hsa-mir-1915 MI0008336UGAGAGGCCGCACCUUGCCUUGCUGCCCGGGCCGUGCACCCGUGGGCCCCAGGGCGACGCGGCGGGGGCGGCCCUAGCGA> hsa-mir-1972-1 MI0009982UAUAGGCAUGUGCCACCACACCUGGCUUAAAUGUGUCAUUUAAAAAUUCAGGCCAGGCACAGUGGCUCAUGCCUGUA> hsa-mir-1972-2 MI0015977UAUAGGCAUGUGCCACCACACCUGGCUUAAAUGUGUCAUUUAAAAAUUCAGGCCAGGCACAGUGGCUCAUGCCUGUA> hsa- mir-1973 MI0009983UAUGUUCAACGGCCAUGGUAUCCUGACCGUGCAAAGGUAGCAUA> hsa-mir-1976 MI0009986GCAGCAAGGAAGGCAGGGGUCCUAAGGUGUGUCCUCCUGCCCUCCUUGCUGU> hsa-mir-2052 MI0010486CUGUUUUGAU CUUUAAGUGUUAAUUAAACCUCUAUUUACAUAGCAAG> hsa-mir-2054 MI0010488CUGUAAUAUAAAUUUAAUUUAUUCUCUAUCAUUAAAAAAUGUAUUACAG> hsa-mir-2110 MI0010629CAGGGGUUUGGGGAAACGGCCGCUGAGUGAGGCGUCGGCUGUGUUUCUCACCGCGGUCUUUUCCUCCCACUCUUG> hsa-mir-2113 MI0003939UUUUCAAAGCAAUGUGUGACAGGUACAGGGACAAAUCCCGUUAAUAAGUAAGAGGAUUUGUGCUUGGCUCUGUCACAUGCCACUUUGAAAA> hsa-mir-2114 MI0010633CCUCCAUGCUCCUAGUCCCUUCCUUGAAGCGGUCGGAUAAUCACAUGACGAGCCUCAAGCAAGGGACUUCAAGCUGGUGG> hsa-mir-2115 MI0010634ACUGUCAUCCCACUGCUUCCAGCUUCCAUGACUCCUGAUGGAGGAAUCACAUGAAUUCAUCAGAAUUCAUGGAGGCUAGAAGCAGUAUGAGGAUCAUUUA> hsa-mir-2116 MI0010635GACCUAGGCUAGGGGUUCUUAGCAUAGGAGGUCUUCCCAUGCUAAGAAGUCCUCCCAUGCCAAGAACUCCCAGACUAGGA> hsa-mir-2117 MI0010636GCUCUGAUUUACUUCUGUCCGGCAUGGUGAACAGCAGGAUUGGCUGUAGCUGUUCUCUUUGCCAAGGACAGAUCUGAUCU > hsa-mir-2276 MI0011282GUGUUCUUCCAGUCCGCUCUGUCACCUUGCAGACGGCUUUCUCUCCGAAUGUCUGCAAGUGUCAGAGGCGAGGAGUGGCAGCUGCAU> hsa-mir-2277 MI0011284GUGCUUCCUGCGGAGCGCGGGCUGAGCUGCC 1285GUGCUGCAGGUGUUGGAGAGCAGUGUGUGUUGCCUGGGGACUGUGUGGACUGGUAUCACCCAGACAGCUUGCACUGACUCCAGACCCUGCCGUCAU> hsa-mir-2355 MI0015873CAGACGUGUCAUCCCCAGAUACAAUGGACAAUAUGCUAUUAUAAUCGUAUGGCAUUGUCCUUGCUGUUUGGAGAUAAUACUGCUGAC> hsa-mir-2392 MI0016870AUGGUCCCUCCCAAUCCAGCCAUUCCUCAGACCAGGUGGCUCCCGAGCCACCCCAGGCUGUAGGAUGGGGGUGAGAGGUGCUAG> hsa-mir-2467 MI0017432GGACAGGCACCUGAGGCUCUGUUAGCCUUGGCUCUGGGUCCUGCUCCUUAGAGCAGAGGCAGAGAGGCUCAGGGUCUGUCU> hsa-mir-2681 MI0012062GCCCCCUUUUCACGCAUUUGUGUUUUACCACCUCCAGGAGACUGCCCAAAGACUCUUCAGUAUCAUGGAGUUGGUAAAGCACAGAUGCAUGAAUAAUUCAACGUG> hsa-mir-2682 MI0012063ACCUUCCUGAAAGAGGUUGGGGCAGGCAGUGACUGUUCAGACGUCCAAUCUCUUUGGGACGCCUCUUCAGCGCUGUCUUCCCUGCCUCUGCCUUUAGGACGAGUCUCAAA> hsa-mir-2861 MI0013006GGCGCCUCUGCAGCUCCGGCUCCCCCUGGCCUCUCGGGAACUACAAGUCCCAGGGGGCCUGGCGGUGGGCGGCGGGCGGAAGAGGCGGGG> hsa-mir-2909 MI0013083GGUGUUAGGGCCAACAUCUCUUGGUCUUUCCCCUGUGGUCCCAAGAUGGCUGUUGCAACUUAACGCCAU > hsa-mir-2964a MI0017299GGAGCUCAGCCACAGAUGUCCAGCCACAAUUCUCGGUUGGCCGCAGACUCGUACAAGAAUGCGUUUGGACAAUCAGUGGCGAAGCCC> hsa-mir-3064 MI0017375GGUCUGGCUGUUGUGGUGUGCAAAACUCCGUACAUUGCUAUUUUGCCACACUGCAACACCUUACAG> hsa-mir-3065 MI0014228CUGCCCUCUUCAACAAAAUCACUGAUGCUGGAGUCGCCUGAGUCAUCACUCAGCACCAGGAUAUUGUUGGAGAGGACAG> hsa-mir-3074 MI0014181GCUCGACUCCUGUUCCUGCUGAACUGAGCCAGUGUGUAAAAUGAGAACUGAUAUCAGCUCAGUAGGCACCGGAGGGCGGGU> hsa-mir-3115 MI0014127UCUGAAUAUGGGUUUACUAGUUGGUGGUGAAUUCAUGAGUCGCCAACUAUUAGGCCUUUAUGUCCAGA> hsa-mir-3116-1 MI0014128CUUUAUUGAGUCCCUACUAUGUUCCAGGCACUGGGUAUCGUAGGUGCCUGGAACAUAGUAGGGACUCAAUAAAG> hsa-mir-3116-2 MI0014129UAUUGAGUCCCUACUAUGUUCCAGGCACCUACGAUACCCAGUGCCUGGAACAUAGUAGGGACUCAAUA> hsa-mir- 3117 MI0014130CCCUAAAGGGCCAGACACUAUACGAGUCAUAUAAGGGAAGGCAUUAUAGGACUCAUAUAGUGCCAGGUGUUUUGUGGG> hsa-mir-3118-1 MI0014131CACACUACAAUAAUUUUCAUAAUGCAAUCACACAUAAUCACUAUGUGACUGCAUUAUGAAAAUUCUUGUAGUGUG> hsa-mir-3118-2 MI0014132ACACUACAAUAAUUUUCAUAAUGCAAUCACACAUAAUCACUAUGUGACUGCAUUAUGAAAAUUCUUGUAGUGU> hsa-mir-3118-3 MI0014133CACACUACAAUAAUUUUCAUAAUGCAAUCACACAUAAUCACUAUGUGACUGCAUUAUGAAAAUUCUUGUA
GUGUG> hsa-mir-3118-4 MI0014207CAUACUACAAUAAUUUUCAUAAUGCAAUCACACACAAUCACCGUGUGACUGCAUUAUGAAAAUUCUUCUAGUGUG> hsa-mir-3118-5 MI0014243CACACAUACAAUAAUAUUCAUAAUGCAAUCACACACAAUCACCAUGUGACUGCAUUAUGAAAAUUCUUCUAGUGUG> hsa-mir-3118-6 MI0015981CAUACUACAAUAAUUUUCAUAAUGCAAUCACACACAAUCACCGUGUGACUGCAUUAUGAAAAUUCUUCUAGUGUG> hsa-mir-3119-1 MI0014134AUUAACUCUGGCUUUUAACUUUGAUGGCAAAGGGGUAGCUAAACAAUCUAUGUCUUUGCCAUCAAAGUUAAAAGCCAUAGUUAAU> hsa-mir-3119-2 MI0014135AUUAACUAUGGCUUUUAACUUUGAUGGCAAAGACAUAGAUUGUUUAGCUACCCCUUUGCCAUCAAAGUUAAAAGCCAGAGUUAAU> hsa-mir -3120 MI0014136GUCAUGUGACUGCCUGUCUGUGCCUGCUGUACAGGUGAGCGGAUGUUCUGCACAGCAAGUGUAGACAGGCAGACACAUGAC> hsa-mir-3121 MI0014137AAAUGGUUAUGUCCUUUGCCUAUUCUAUUUAAGACACCCUGUACCUUAAAUAGAGUAGGCAAAGGACAGAAACAUUU> hsa-mir-3122 MI0014138ACCAGCUCUGUUGGGACAAGAGGACGGUCUUCUUUUGGAAGGAAGACCAUCAUCUUGUCCGAAGAGAGCUGGU> hsa-mir-3123 MI0014139AUGGAUUUGAUUGAAUGAUUCUCCCAUUUCCACAUGGAGAGUGGAGCCCAGAGAAUUGUUUAAUCAUGUAUCCAU> hsa-mir-3124 MI0014140GCGGGCUUCGCGGGCGAAGGCAAAGUCGAUUUCCAAAAGUGACUUUCCUCACU CCCGUGAAGUCGGC> hsa-mir-3125 MI0014142GAGAAUGGGUAGAGGAAGCUGUGGAGAGAACUCACGGUGCCUGUGGUUCGAGAUCCCCGCCUUCCUCCUCCUUUCCUC> hsa-mir-3126 MI0014143AUGAUUAUAUGAGGGACAGAUGCCAGAAGCACUGGUUAUGAUUUGCAUCUGGCAUCCGUCACACAGAUAAUUAU> hsa-mir-3127 MI0014144GGCCAGGCCCAUCAGGGCUUGUGGAAUGGGAAGGAGAAGGGACGCUUCCCCUUCUGCAGGCCUGCUGGGUGUGGCU> hsa-mir-3128 MI0014145UUCCUCUGGCAAGUAAAAAACUCUCAUUUUCCUUAAAAAAUGAGAGUUUUUUACUUGCAAUAGGAA> hsa-mir-3129 MI0014146GUACUUGGGCAGUAGUGUAGAGAUUGGUUUGCCUGUUAAUGAAUUCAAACUAAUCUCUACACUGCUGCCCAAGAGC> hsa-mir-3130-1 MI0014147CUUGUCAUGUCUUACCCAGUCUCCGGUGCAGCCUGUUGUCAAGGCUGCACCGGAGACUGGGUAAGACAUGACAAG> hsa-mir- 3130-2 MI0014148CUUGUCAUGUCUUACCCAGUCUCCGGUGCAGCCUUGACAACAGGCUGCACCGGAGACUGGGUAAGACAUGACAAG> hsa-mir-3131 MI0014151GAGUCGAGGACUGGUGGAAGGGCCUUUCCCCUCAGACCAAGGCCCUGGCCCCAGCUUCUUCUC> hsa-mir-3132 MI0014152GGUGGGAUGGGUAGAGAAGGAGCUCAGAGGACGGUGCGCCUUGUUUCCCUUGAGCCCUCCCUCUCUCAUCCCACC> hsa-mir-3133 MI0014153CAGAAAUUGUAAAGAACUCUUAAAACCCAAUAGUAAAAAGACAACCUGUUGAGUUUUAAGAGUUCUUUAUAUAUUCUG> hsa-mir-3134 MI 0014155UGUAUCCAAUGUGUAGUCUUUUAUCCCUCACAUGGAGUAAAAUAUGAUGGAUAAAAGACUACAUAUUGGGUACA> hsa-mir-3135a MI0014156UCACUUUGGUGCCUAGGCUGAGACUGCAGUGGUGCAAUCUCAGUUCACUGCAGCCUUGACCUCCUGGGCUCAGGUGA> hsa-mir-3135b MI0016809UGCCCAGGCUGGAGCGAGUGCAGUGGUGCAGUCAGUCCUAGCUCACUGCAGCCUCGAACUCCUGGGCU> hsa-mir-3136 MI0014158AAUAUGAAACUGACUGAAUAGGUAGGGUCAUUUUUCUGUGACUGCACAUGGCCCAACCUAUUCAGUUAGUUCCAUAUU> hsa-mir-3137 MI0014160UACAGGUCUGUAGCCUGGGAGCAAUGGGGUGUAUGGUAUAGGGGUAGCCUCGUGCUCCUGGGCUACAAACCUGUA> hsa-mir-3138 MI0014161CCCUCCUCGGCACUUCCCCCACCUCACUGCCCGGGUGCCCACAAGACUGUGGACAGUGAGGUAGAGGGAGUGCCGAGGAGGG> hsa-mir-3139 MI0014162GGCUCAGAGUAGGAGCUCAACAGAUGCCUGUUGACUGAAUAAUAAACAGGUAUCGCAGGAGCUUUUGUUAUGUGCC> hsa-mir-3140 MI0014163CCUCUUGAGGUACCUGAAUUACCAAAAGCUUUAUGUAUUCUGAAGUUAUUGAAAAUAAGAGCUUUUGGGAAUUCAGGUAGUUCAGGAGUG > hsa-mir-3141 MI0014165UCACCCGGUGAGGGCGGGUGGAGGAGGAGGGUCCCCACCAUCAGCCUUCACUGGGACGGGA> hsa-mir-3142 MI0014166UUCAGAAAGGCCUUUCUGAACCUUCAGAAAGGCUGCUGAAUCUAGAAAGGCCUUUCUGAACCUAGAAAGGC 67UAGAUAACAUUGUAAAGCGCUUCUUUCGCGGUUGGGCUGGAGCAACUCUUUACAAUGUUUCUA> hsa-mir-3144 MI0014169AACUACACUUUAAGGGGACCAAAGAGAUAUAUAGAUAUCAGCUACCUAUAUACCUGUUCGGUCUCUUUAAAGUGUAGUU> hsa-mir-3145 MI0014170UAUAUGAGUUCAACUCCAAACACUCAAAACUCAUUGUUGAAUGGAAUGAGAUAUUUUGAGUGUUUGGAAUUGAACUCGUAUA> hsa-mir-3146 MI0014172GCUAAGUCCCUUCUUUCUAUCCUAGUAUAACUUGAAGAAUUCAAAUAGUCAUGCUAGGAUAGAAAGAAUGGGACUUGGC> hsa-mir-3147 MI0014173GUCCGGGUUGGGCAGUGAGGAGGGUGUGACGCCGCGAAGUGCACCUCGCCCUUGUCCAACUCGGAC> hsa-mir-3148 MI0014175GAGUUAAGAUGGAAAAAACUGGUGUGUGCUUAUUGAUGUAGCCAACAAGCAUACAUCAGUUUUUUCCAACUUAACUC> hsa-mir-3149 MI0014176AUACAUACAUGUACACACACAUGUCAUCCACACACAUACAUAUAUAUAUGUUUGUAUGGAUAUGUGUGUGUAUGUGUGUGUAU> hsa-mir-3150a MI0014177GGGAAGCAGGCCAACCUCGACGAUCUCCUCAGCACCUGAACGCCAAGGCUGGGGAGAUCCUCGAGGUUGGCCUGCUUUCC > hsa-mir-3150b MI0016426GAGGGAAAGCAGGCCAACCUCGAGGAUCUCCCCAGCCUUGGCGUUCAGGUGCUGAGGAGAUCGUCGAGGUUGGCCUGCUUCCCCUC> hsa-mir-3151 MI0014178GGGGUGAUGGGUGGGGCAAUGGGAUCAGGUGCCUCAAAGGGCAUCCC. 14179GUGCAGAGUUAUUGCCUCUGUUCUAACACAAGACUAGGCUUCCCUGUGUUAGAAUAGGGGCAAUAACUCUGCAC> hsa-mir-3153 MI0014180GACAAAUUUUAAAUGUCCCUGUCCCCUUCCCCCCAAUUAAAGUAGAUUGGGGGAAAGCGAGUAGGGACAUUUAAAAUUUGUU> hsa-mir-3154 MI0014182GGCCCCUCCUUCUCAGCCCCAGCUCCCGCUCACCCCUGCCACGUCAAAGGAGGCAGAAGGGGAGUUGGGAGCAGAGAGGGGACC> hsa-mir-3155a MI0014183UCCGGGCAUCACCUCCCACUGCAGAGCCUGGGGAGCCGGACAGCUCCCUUCCCAGGCUCUGCAGUGGGAACUGAUGCCUGGA> hsa-mir-3155b MI0016839CCACUGCAGAGCCUGGGAAGGGAGCUGUCCGGCUCCCCAGGCUCUGCAGUGGGAGU> hsa-mir-3156-1 MI0014184GCAGAAGAAAGAUCUGGAAGUGGGAGACACUUUUACUAUAUAUAGUGGCUCCCACUUCCAGAUCUUUCUCUCUGU> hsa-mir-3156-2 MI0014230UGCAGAAGAAAGAUCUGGAAGUGGGAGACACUUUCACUAUAUAUAGUGGCUCCCACUUCCAGAUCUUUCUCUCUGUA> hsa- mir-3156-3 MI0014242UGCAGAAGAAAGAUCUGGAAGUGGGAGACACUUUCACUAUAUAUAGUGGCUCCCACUUCCUGAUCUUUCUCUCUGUA> hsa-mir-3157 MI0014185GGGAAGGGCUUCAGCCAGGCUAGUGCAGUCUGCUUUGUGCCAACACUGGGGUGAUGACUGCCCUAGUCUAGCUGAAGCUUUUCCC> hsa-mir-3158-1 MI0014186AUUCAGGCCGGUCCUGCAGAGAGGAAGCCCUUCUGCUUACAGGUAUUGGAAGGGCUUCCUCUCUGCAGGACCGGCCUGAAU> hsa- mir-3158-2 MI0014187AUUCAGGCCGGUCCUGCAGAGAGGAAGCCCUUCCAAUACCUGUAAGCAGAAGGGCUUCCUCUCUGCAGGACCGGCCUGAAU> hsa-mir-3159 MI0014188CCAAAGUCCUAGGAUUACAAGUGUCGGCCACGGGCUGGGCACAGUGGCUCACGCCUGUAAUCCCAGCAUUUUGG> hsa-mir-3160-1 MI0014189GGACCUGCCCUGGGCUUUCUAGUCUCAGCUCUCCUCCAGCUCAGCUGGUCAGGAGAGCUGAGACUAGAAAGCCCAGGGCAGGUUC> hsa-mir-3160-2 MI0014190ACCUGCCCUGGGCUUUCUAGUCUCAGCUCUCCUGACCAGCUGAGCUGGAGGAGAGCUGAGACUAGAAAGCCCAGGGCAGGU> hsa-mir-3161 MI0014191CCUCGAGAGCUGAUAAGAACAGAGGCCCAGAUUGAAGUUGAAUAGUGCUGGGCCUUUGUUUUUACCAAGUUCCCUGG> hsa-mir-3162 MI0014192CUGACUUUUUUAGGGAGUAGAAGGGUGGGGAGCAUGAACAAUGUUUCUCACUCCCUACCCCUCCACUCCCCAAAAAAGUCAG> hsa-mir- 3163 MI0014193UUCCUCAUCUAUAAAAUGAGGGCAGUAAGACCUUCCUUCCUUGUCUUACUACCCCCAUUUUAUAGAUGAGGAA> hsa-mir-3164 MI0014194CUUGGAAACUGUGACUUUAAGGGAAAUGGCGCACAGCAGACCCUGCAAUCAUGCCGUUUUGCUUGAAGUCGCAGUUUCCCAGG> hsa-mir-3165 MI0014195CCGGUGGCAAGGUGGAUGCAAUGUGACCUCAACUCUUGGUCCUCUGAGGUCACAUUGUAUCCACCUUACCACUGG> hsa-mir-3166 MI0014196AAAUUUUUUUGAGGCCAGUAGGCAUUGUCUGCGUUAGGAUUUCUGUAUCAUCCUCCUA ACGCAGACAAUGCCUACUGGCCUAAGAAAAAUUU> hsa-mir-3167 MI0014198GGCUGUGGAGGCACCAGUAUUUCUGAAAUUCUUUUUUCUGAAAUUCUUCAGGAAGGAUUUCAGAAAUACUGGUGUCCCGACAGCC> hsa-mir-3168 MI0014199AAGAUCAUGAGUUCUACAGUCAGACAGCCUGAGUUGGAGGCUCAUCUUCACUUCUUGCUGUGUGACCCUGGGCCAGUGACUU> hsa-mir-3169 MI0014200AUGUGAAAACAUAGGACUGUGCUUGGCACAUAGCACAAAGUCUUAUGGUACUGUGUGCCAAGCAUAGUCCUGUGUUUUUACAU> hsa-mir-3170 MI0014201CUGGUAACACUGGGGUUCUGAGACAGACAGUGUUAGCUCCAGAAGCAUUGCCUGUCUUAGAACCCCUAUGUUACCAG> hsa-mir-3171 MI0014202UAUAUAUAGAGAUGUAUGGAAUCUGUAUAUAUCUAUAUAUAUGUGUAUAUAUAGAUUCCAUAAAUCUAUAUAUG> hsa-mir-3173 MI0014204UCCCUGCCCUGCCUGUUUUCUCCUUUGUGAUUUUAUGAGAACAAAGGAGGAAAUAGGCAGGCCAGGGA> hsa-mir-3174 MI0014208GUUACCUGGUAGUGAGUUAGAGAUGCAGAGCCCUGGGCUCCUCAGCAAACCUACUGGAUCUGCAUUUUAAUUCACAUGCAUGGUAAU > hsa-mir-3175 MI0014209CCUGGGGGGCGGGGAGAGAACGCAGUGACGUCUGGCCGCGUGCGCAUGUCGGGCGCUUUCCUCCCCCUACCCAGG> hsa-mir-3176 MI0014210UGGCCUCUCCAGUCUGCAGCUCCCGGCAGCCUCGGCACACUCUCCCGGGC GUACACACGUGCCAGGCGCUGUCUUGAGACAUUCGCGCAGUGCACGGCACUGGGGACACGUGGCACUGG> hsa-mir-3178 MI0014212GAGGCUGGGCGGGGCGCGGCCGGAUCGGUCGAGAGCGUCCUGGCUGAUGACGGUCUCCCGUGCCCACGCCCCAAACGCAGUCUC> hsa-mir-3179-1 MI0014213CAGGAUCACAGACGUUUAAAUUACACUCCUUCUGCUGUGCCUUACAGCAGUAGAAGGGGUGAAAUUUAAACGUCUGUGAUCCUG> hsa-mir-3179-2 MI0014216CAGGAUCACAGACGUUUAAAUUACACUCCUUCUGCUGUGCCUUACAGCAGUAGAAGGGGUGAAAUUUAAACGUCUGUGAUCCUG> hsa-mir-3179-3 MI0014221CAGGAUCACAGACGUUUAAAUUACACUCCUUCUGCUGUGCCUUACAGCAGUAGAAGGGGUGAAAUUUAAACGUCUGUGAUCCUG> hsa-mir-3180-1 MI0014214CAGUGCGACGGGCGGAGCUUCCAGACGCUCCGCCCCACGUCGCAUGCGCCCCGGGAAAGCGUGGGGCGGAGCUUCCGGAGGCCCCGCCCUGCUG> hsa-mir-3180 -2 MI0014215GCGACGGGCGGAGCUUCCAGACGCUCCGCCCCACGUCGCAUGCGCCCCGGGAAAGCGUGGGGCGGAGCUUCCGGAGGCCCCGCCCUGC> hsa-mir-3180-3 MI0014217CAGUGCGACGGGCGGAGCUUCCAGACGCUCCGCCCCACGUCGCAUGCGCCCCGGGAAAGCGUGGGGCGGAGCUUCCGGAGGCCCCGCCCUGCUG> hsa-mir-3180-4 MI0016408GCUCCGCCCCACGUCGCAUGCGCCCCGGGAACGCGUGGGGCGGAGCUUCCGGAGGCCCCGCUCUGCUGCCGACCCUGUGGAGCGGAGGGUGAAGCCUCCGGAUGCCAGUCCCUCAUCGCUG GCCUGGUCGCGCUGUGGCGAAGGGGGCGGAGC> hsa-mir-3180-5 MI0016409GCUCCGCCCCACGUCGCAUGCGCCCCGGGAACGCGUGGGGCGGAGCUUCCGGAGGCCCCGCCCUGCUGCCGACCCUGUGGAGCGGAGGGUGAAGCCUCCGGAUGCCAGUCCCUCAUCGCUGGCCCGGUCGCGCUGUGGCGAAGGGGGCGGAGC> hsa-mir-3181 MI0014223CGGCGACCAUCGGGCCCUCGGCGCCGGCCCGUUAGUUGCCCGGGCCCGAGCCGGCCGGGCCCGCGGGUUGCCG> hsa-mir-3182 MI0014224GCUGCUUCUGUAGUGUAGUCCGUGCAUCCGCCCUUCGAUGCUUGGGUUGGAUCAUAGAGCAGU> hsa-mir-3183 MI0014225CUCUGCCCUGCCUCUCUCGGAGUCGCUCGGAGCAGUCACGUUGACGGAAUCCUCCGGCGCCUCCUCGAGGGAGGAGAGGCAGGG> hsa-mir-3184 MI0014226AAGCAAGACUGAGGGGCCUCAGACCGAGCUUUUGGAAAAUAGAAAAGUCUCGCUCUCUGCCCCUCAGCCUAACUU> hsa-mir-3185 MI0014227GAAUGGAAGAAGAAGGCGGUCGGUCUGCGGGAGCCAGGCCGCAGAGCCAUCCGCCUUCUGUCCAUGUC> hsa-mir- 3186 MI0014229AGCCUGCGGUUCCAACAGGCGUCUGUCUACGUGGCUUCAACCAAGUUCAAAGUCACGCGGAGAGAUGGCUUUGGAACCAGGGGCU> hsa-mir-3187 MI0014231GCUGGCCCUGGGCAGCGUGUGGCUGAAGGUCACCAUGUUCUCCUUGGCCAUGGGGCUGCGCGGGGCCAGC> hsa-mir-3188 MI0014232GGCGCCUCCUGCUCUGCUGUGCCGCCAGGGCCUCCCCUAGCGCGCCUUCUGGAGAGGCUUUGUGCGGAUACGGGGCUGGAGGCCU> hs a-mir-3189 MI0014233GCCUCAGUUGCCCCAUCUGUGCCCUGGGUAGGAAUAUCCUGGAUCCCCUUGGGUCUGAUGGGGUAGCCGAUGC> hsa-mir-3190 MI0014235CUGGGGUCACCUGUCUGGCCAGCUACGUCCCCACGGCCCUUGUCAGUGUGGAAGGUAGACGGCCAGAGAGGUGACCCCGG> hsa-mir-3191 MI0014236GGGGUCACCUCUCUGGCCGUCUACCUUCCACACUGACAAGGGCCGUGGGGACGUAGCUGGCCAGACAGGUGACCCC> hsa-mir-3192 MI0014237GGAAGGGAUUCUGGGAGGUUGUAGCAGUGGAAAAAGUUCUUUUCUUCCUCUGAUCGCCCUCUCAGCUCUUUCCUUCU> hsa-mir-3193 MI0014238UCCUGCGUAGGAUCUGAGGAGUGGACGAGUCUCAUUACCCAGCUCCUGAGCAGGA> hsa-mir-3194 MI0014239AGGUGGCAGGGCCAGCCACCAGGAGGGCUGCGUGCCACCCGGGCAGCUCUGCUGCUCACUGGCAGUGUCACCU> hsa-mir-3195 MI0014240CCGCAGCCGCCGCGCCGGGCCCGGGUUGGCCGCUGACCCCCGCGGGGCCCCCGGCGGCCGGGGCGGGGGCGGGGGCUGCCCCGG> hsa -mir-3196 MI0014241GGGUGGGGGCGGGGCGGCAGGGGCCUCCCCCAGUGCCAGGCCCCAUUCUGCUUCUCCCAGCU> hsa-mir-3197 MI0014245GGCGAGGGGAGGCGCAGGCUCGGAAAGGCGCGCGAGGCUCCAGGCUCCUUCCCGAUCCACCGCUCUCUCCUUG
GAGAGAGCACAGUC> hsa-mir-3198-2 MI0017335GACUCUGCUCUCACUGUUCACCCAGCACUAGCAGUACCAGAUGGUUCUGUGGAGUCCUGGGGAAUGGAGAGAGCACAGUC> hsa-mir-3199-1 MI0014247GGUGACUCCAGGGACUGCCUUAGGAGAAAGUUUCUGGAAGUUCUGACAUUCCAGAAACUUUCUCCUAAGGCAGUCCCUGGGAGUCACU> hsa-mir-3199-2 MI0014248GUGACUCCCAGGGACUGCCUUAGGAGAAAGUUUCUGGAAUGUCAGAACUUCCAGAAACUUUCUCCUAAGGCAGUCCCUGGAGUCAC> hsa-mir-3200 MI0014249GGUGGUCGAGGGAAUCUGAGAAGGCGCACAAGGUUUGUGUCCAAUACAGUCCACACCUUGCGCUACUCAGGUCUGCUCGUGCCCU> hsa-mir-3201 MI0014250GGGAUAUGAAGAAAAAUAAGAGGCUAGGAUUGCCUCUUAUUUUUACAUGCCC> hsa-mir-3202-1 MI0014252UAUUAAUAUGGAAGGGAGAAGAGCUUUAAUGAUUGGAGUCAUUUUCAGAGCAUUAAAGCUCUUCUCCCUUCCAUAUUAAUG> hsa-mir-3202-2 MI0014253AUUAAUAUGGAAGGGAGAAGAGCUUUAAUGCUCUGAAAAUGACUCCAAUCAUUAAAGCUCUUCUCCCUUCCAUAUUAAU> hsa-mir-3529 MI0017351GGCACCAUUAGGUAGACUGGGAUUUGUUGUUGAGCGCAGUAAGACAACAACAAAAUCACUAGUCUUCCAGAUGGGGCC> hsa-mir-3545 MI0017343GCGCCCGCCGGGUCUAGUGGUCCUAAACAUUUCACAAUUGCGCUACAGAACUGUUGAACUGUUAAGAACCACUGGACCCAGCGCGC> hsa-mir-3591 MI0017383CAGUAGCUAUUUAGUGUGAUAAUGGCGUUUG AUAGUUUAGACACAAACACCAUUGUCACACUCCACAGCUCUG> hsa-mir-3605 MI0015995ACUUUAUACGUGUAAUUGUGAUGAGGAUGGAUAGCAAGGAAGCCGCUCCCACCUGACCCUCACGGCCUCCGUGUUACCUGUCCUCUAGGUGGGACGCUCG> hsa-mir-3606 MI0015996UUGUUGCUAUCUAGGUUAGUGAAGGCUAUUUUAAUUUUUUUAAAAUUUCUUUCACUACUUAGG> hsa-mir-3607 MI0015997AAGGUUGCGGUGCAUGUGAUGAAGCAAAUCAGUAUGAAUGAAUUCAUGAUACUGUAAACGCUUUCUGAUGUACUACUCA> hsa-mir-3609 MI0015999GUAACAGUAACUUUUAUUCUCAUUUUCCUUUUCUCUACCUUGUAGAGAAGCAAAGUGAUGAGUAAUACUGGCUGGAGCCC> hsa-mir-3610 MI0016000AAGAGCCGCGGCGUAACGGCAGCCAUCUUGUUUGUUUGAGUGAAUCGGAAAGGAGGCGCCGGCUGUGGCGGCG> hsa-mir-3611 MI0016001AGCAGGUCUAAUAAGAAUUUCUUUUUCUUCACAAUUAUGAAAGAAAAGAAAUUGUGAAGAAAGAAAUUCUUACUAGUUUUGCU> hsa-mir-3612 MI0016002GGGACUGGGGAUGAGGAGGCAUCUUGAGAAAUGGAAGGAAUGGGAUCUACUUCCAGUUCACUAGAGGCGUCCUGACACCCCUAGCUC > hsa-mir-3613 MI0016003UGGUUGGGUUUGGAUUGUUGUACUUUUUUUUUGUUCGUUGCAUUUUAGGAACAAAAAAAAAAGCCCAACCCUUCACACCACUUCA> hsa-mir-3614 MI0016004GGUUCUGUCUUGGGCCACUUG MI0016005GACUCUGGGACGCUCAGACGCCGCGCGGGGCGGGGAUUGGUCUGUGGUCCUCUCUCGGCUCCUCGCGGCUCGCGGCGGCCGACGGUU> hsa-mir-3616 MI0016006UGUCACUCCGCCAGCAUCAUGAAGUGCACUCAUGAUAUGUUUGCCCCAUCAGCGUGUCACGAGGGCAUUUCAUGAUGCAGGCGGGGUUGGCA> hsa-mir-3617 MI0016007AGGUCAUAGAAAGACAUAGUUGCAAGAUGGGAUUAGAAACCAUAUGUCUCAUCAGCACCCUAUGUCCUUUCUCUGCCCU> hsa-mir-3618 MI0016008UAAGCUGAGUGCAUUGUGAUUUCCAAUAAUUGAGGCAGUGGUUCUAAAAGCUGUCUACAUUAAUGAAAAGAGCAAUGUGGCCAGCUUG> hsa-mir-3619 MI0016009ACGGCAUCUUUGCACUCAGCAGGCAGGCUGGUGCAGCCCGUGGUGGGGGACCAUCCUGCCUGCUGUGGGGUAAGGACGGCUGU> hsa-mir-3620 MI0016011GUGAGGUGGGGGCCAGCAGGGAGUGGGCUGGGCUGGGCUGGGCCAAGGUACAAGGCCUCACCCUGCAUCCCGCACCCAG> hsa-mir-3621 MI0016012GUGAGCUGCUGGGGACGCGGGUCGGGGUCUGCAGGGCGGUGCGGCAGCCGCCACCUGACGCCGCGCCUUUGUCUGUGUCCCACAG> hsa-mir-3622a MI0016013AAUAGAGGGUGCACAGGCACGGGAGCUCAGGUGAGGCAGGGAGCUGAGCUCACCUGACCUCCCAUGCCUGUGCACCCUCUAUU > hsa-mir-3622b MI0016014AGUGAUAUAAUAGAGGGUGCACAGGCAUGGGAGGUCAGGUGAGCUCAGCUCCCUGCCUCACCUGCUCCCGUGCCUGGCCUCUAUUGGCU> hsa-mir-3646 MI0016046UUCAGUAGGU UGGGUUCAUUUCAUUUUCAUGACAACCCUAUAUGGGAAAAUGUUGUGAAAAUGAAAUGAGCCCAGCCCAUUGAA> hsa-mir-3648 MI0016048CGCGACUGCGGCGGCGGUGGUGGGGGGAGCCGCGGGGAUCGCCGAGGGCCGGUCGGCCGCCCCGGGUGCCGCGCGGUGCCGCCGGCGGCGGUGAGGCCCCGCGCGUGUGUCCCGGCUGCGGUCGGCCGCGCUCGAGGGGUCCCCGUGGCGUCCCCUUCCCCGCCGGCCGCCUUUCUCGCG> hsa-mir-3649 MI0016049GCUUGGAACAGGCACCUGUGUGUGCCCAAGUGUUUCUAGCAAACACAGGGACCUGAGUGUCUAAGC> hsa-mir-3650 MI0016050UCAAGGUGUGUCUGUAGAGUCCUGACUGCGUGCCAGGGGCUCUGUCUGGCACAUUUCUGA> hsa-mir-3651 MI0016051GAUUCGAUGGGCCAUAGCAAUCCUGUGAUUUAUGCAUGGAGGCUGCUUCUCCUCAGCAGCUGCCAUAGCCCGGUCGCUGGUACAUGAUUC> hsa-mir-3652 MI0016052CGGCUGGAGGUGUGAGGAUCCGAACCCAGGGGUGGGGGGUGGAGGCGGCUCCUGCGAUCGAAGGGGACUUGAGACUCACCGGCCGCACGCCAUGAGGGCCCUGUGGGUGCUGGGCCUCUGCUGCGUCCUGC> hsa-mir-3653 MI0016053UCCCUGGGGACCCCUGGCAGCCCCUCCUGAUGAUUCUUCUUCCUGAGCACGCUCAUGAUGAGCAAACUGAGCCUCUAAGAAGUUGACUGAAGGGGCUGCUUCCCCAAGGA> hsa-mir-3654 MI0016054UUCAUGAGCUGCAAUCUCAUCACUGGAAUGUUCCAGCGACUGGACAAGCUGAGGAA > hsa-mir-3655 MI0016055GCUUGUCGCUGCGGUGUUGCUGUUGGAGACUCGAUUGUUGGUGACAGCG AAAGAACGAUAACAAAAUGCCGGAGCGAGAUAGU> hsa-mir-3656 MI0016056CUUUCGGCCAGCGGGACGGCAUCCGAGGUGGGCUAGGCUCGGGCCCGUGGCGGGUGCGGGGGUGGGAGG> hsa-mir-3657 MI0016057UGUGUCCCAUAAUUAAAUAAUGAAAUCUGAAAUCACCAAUAAUGGGACACUAAUGUGAUUAAUGUUGUUGUGUCCCAUUAUUGGUGAUUUCAGAUUUCAUAUAUGAUUAAGGACAUA> hsa-mir-3658 MI0016058UAUUUAAGAAAACACCAUGGAGAUGAAAUGCCUUUGAUUUUUUUUUUCUUUUUGUA> hsa-mir-3659 MI0016060UCUACAAGCAGAUACAAGGAUGCCCUUGUACACAACACACGUGCUGCUUGUAUAGACAUGAGUGUUGUCUACGAGGGCAUCCUUGUGUCUGUGUGUGUG> hsa-mir-3660 MI0016061GAAAGAAGAACUGGACAAAAUUAAAAUGCUCUUCUGUCAUUGUAAUAGUUCAUAUGGGCACUGACAGGAGAGCAUUUUGACUUUGUCAAGUGUGUCUGCU> hsa-mir-3661 MI0016062CACCUUCUCGCAGAGGCUCUUGACCUGGGACUCGGACAGCUGCUUGCACUCGUUCAGCUGCUCGAUCCACUGGUCCAGCUCCUUGGUGAACACCUU> hsa-mir-3662 MI0016063UGUGUUUUCCUCAACGCUCACAGUUACACUUCUUACUCUCAAUCCAUUCAUAUUGAAAAUGAUGAGUAGUGACUGAUGAAGCACAAAUCAGCCAA > hsa-mir-3663 MI0016064CCCGGGACCUUGGUCCAGGCGCUGGUCUGCGUGGUGCUCGGGUGGAUAAGUCUGAUCUGAGCACCACACAGGCCGGGCGCCGGGACCAAGGGGGCUC> hsa-mir-3664 MI0016065CUGUAAACUUGAAGGUAGGGAACUCUGUCU UCACUCAUGAGUACCUUCCAACACGAGCUCUCAGGAGUAAAGACAGAGUUCCCUACCUUCAAUGUGGAU> hsa-mir-3665 MI0016066GCGGGCGGCGGCGGCGGCAGCAGCAGCAGGUGCGGGGCGGCGGCCGCGCUGGCCGCUCGACUCCGCAGCUGCUCGUUCUGCUUCUCCAGCUUGCGCACCAGCUCC> hsa-mir-3666 MI0016067AGUAAGGUCCGUCAGUUGUAAUGAGACCCAGUGCAAGUGUAGAUGCCGACUCCGUGGCAGAGUUCAGCGUUUCACACUGCCUGGUCUCUGUCACUCUAUUGAAUUAGAUUG> hsa-mir-3667 MI0016068UGAGGAUGAAAGACCCAUUGAGGAGAAGGUUCUGCUGGCUGAGAACCUUCCUCUCCAUGGGUCUUUCAUCCUCA> hsa-mir-3668 MI0016069AUAUAUGAAAUGUAGAGAUUGAUCAAAAUAGUUUCUAUCAAAAUAGUUUUGAUCAAUCUCUGCAAUUUUAUAUAU> hsa-mir-3669 MI0016070AUAUAUAUAUACGGAAUAUAUAUACGGAAUAUAUAUAUACGGAAUAUAUAUAUACGGAAUAUGUAUACGGAAUAUAUAUAU> hsa-mir-3670-1 MI0016071UCUAGACUGGUAUAGCUGCUUUUGGAGCCUCACCUGCUGAGAGCUCACAGCUGUCCUUCUCUAGA> hsa-mir- 3670-2 MI0019112UCUAGACUGGUAUAGCUGCUUUGGAGCCUCACCUGCUGAGAGCUCACAGCUGUCCUUCUCUAGA> hsa-mir-3671 MI0016072AUGUUAUUGCUGCUGCUGUCACAUUUACAUGAAAAUAAUGUAAAUUAUUUUAUUCUA UAUGAGUCUCAUGAUAAUCACAAAGA> hsa-mir-3673 MI0016074AUAUAUAUAUAUGGAAUGUAUAUACGGAAUAUAUAUAUAUAUGGAAUGUAUAUACGGAAUAUAUAUAUAUAUGGAAUGUAUAUACGGAAUAUAUAUAUAUAU> hsa-mir-3674 MI0016075ACAUCACUAUUGUAGAACCUAAGAUUGGCCGUUUGAGAUGUCCUUUCAAGUUUUUGCAUUUCUGAUGU> hsa-mir-3675 MI0016076GGAUGAUAAGUUAUGGGGCUUCUGUAGAGAUUUCUAUGAGAACAUCUCUAAGGAACUCCCCCAAACUGAAUUC> hsa-mir-3676 MI0016077UUGGUUAAAGCGCCUGUCUAGUAAACAGGAGAUCCUGGGUUCGAAUCCCAGCGGUGCCUCCGUGUUUCCCCCACGCUUUUGCCAA> hsa-mir-3677 MI0016078GGCAGUGGCCAGAGCCCUGCAGUGCUGGGCAUGGGCUUCUCGUGGGCUCUGGCCACGGCC> hsa-mir-3678 MI0016079GAAUCCGGUCCGUACAAACUCUGCUGUGUUGAAUGAUUGGUGAGUUUGUUUGCUCAUUGAUUGAAUCACUGCAGAGUUUGUACGGACCGGAUUC> hsa-mir-3679 MI0016080CGUGGUGAGGAUAUGGCAGGGAAGGGGAGUUUCCCUCUAUUCCCUUCCCCCCAGUAAUCUUCAUCAUG > hsa-mir-3680-1 MI0016081AAAUUUAAGGAGGGACUCACUCACAGGAUUGUGCAAAUGCAAAGUUGGCUUUGCAUGACCCUGGGAGUAGGUGCCUCCUUAAAUUU> hsa-mir-3680-2 MI0019113AAAUUUAAGGAGGGACUCACUCACAGGAUUGUGCAAAUGCA GAUGCACUCUGUGCAGGGCCAACUGUGCACACAGUGCUUCAUCCACUACUGGAAGU> hsa-mir-3682 MI0016083UAAGUUAUAUAUGUCUACUUCUACCUGUGUUAUCAUAAUAAAGGUGUCAUGAUGAUACAGGUGGAGGUAGAAAUAUAUAACUUA> hsa-mir-3683 MI0016084GGGUGUACACCCCCUGCGACAUUGGAAGUAGUAUCAUCUCUCCCUUGGAUGCUACGAACAAUAUCACAGAAGGUGUACACCC> hsa-mir-3684 MI0016085AAUCUAAAGGACCUGUACUAGGUUUAACAUGUUGAGCAUUACUCAUGUUAGACCUAGUACACGUCCUUUAGAUU> hsa-mir-3685 MI0016086GUACAUUUCCUACCCUACCUGAAGACUUGAGAUUAUAGUCUUUGGGGGGAUGGGCAAAGUAC> hsa-mir-3686 MI0016087CUCACCUCAUUCAUUUACCUUCUCUUACAGAUCACUUUUCUGCACUGGACAGUGAUCUGUAAGAGAAAGUAAAUGAAAGAGGUGAG> hsa-mir-3687 MI0016088CGCGCGUGCGCCCGAGCGCGGCCCGGUGGUCCCUCCCGGACAGGCGUUCGUGCGACGUGUG> hsa-mir-3688- 1 MI0016089UCUUCACUUUCAAGAGUGGCAAAGUCUUUCCAUAUGUAUGUAUGUAUGUCUGUUACACAUAUGGAAAGACUUUGCCACUCUUUAAAGUGAAGA> hsa-mir-3688-2 MI0017447UCACUUUAAAGAGUGGCAAAGUCUUUCCAUAUGUGUAACAGACAUACAUACAUACAUAUGGAAAGACUUUGCCACUCUUGAAAGUGA> hsa-mir-3689a MI0016090CCUGGGAGGUGUGAUAUCAUGGUUCCUGGGAGGUGUGAUCCUGUGCUUCCUGGGAGGUGUGAUAUCGUGGUUCCUGGG> hsa-mir-3689b MI0016411G AUCCUGUGCUCCCUGGGGGGUCUGAUCCUGUGCUUCCUGGGAGGUGUGAUAUCAUGGUUCCUGGGAGGUGUGAUCCCGUGCUUCCUGGGAGGUGUGAUAUUGUGGUUCCUGGGAGGUGUGAUCCCGUGCUCCCUGGGAGGUGUGAUC> hsa-mir-3689c MI0016832GGGAGGUGUGAUAUCGUGGUUCCUGGGAGGUGUGAUAUCGUGGUUCCUGGGAGGUGUGAUAUUGUGGUUCCU> hsa-mir-3689d-1 MI0016834UGGGAGGUGUGAUCUCACACUCGCUGGGAGGUGUGCUAUCGUCUUCCCCGGGAGGUGUGAUCCUGUUCUUCCUG> hsa-mir-3689d-2 MI0016835ACUGGGAGGUGUGAUCUCACACUCGCUGGGAGGUGUGCUAUCGUCUUCCCUGGGAGGUGUGAUCCUGUUCUUCCUGAGCG> hsa-mir-3689e MI0016836GGGAGGUGUGAUAUCAUGGUUCCUGGGAGGUAUGAUAUCGUGGUUCCUGGGAGGUGUGAUCCCGUGCUCCCU> hsa-mir-3689f MI0016837AGGUGUGAUAUCGUGCUUCCUGGGACGUGUGAUGCUGUGCUUCCUGGGAGGUGUGAUCCCACACUC> hsa-mir-3690 MI0016091CCCAUCUCCACCUGGACCCAGCGUAGACAAAGAGGUGUUUCUACUCCAUAUCUACCUGGACCCAGUGUAGAUGGG> hsa- mir-3691 MI0016092UUGAGGCACUGGGUAGUGGAUGAUGGAGACUCGGUACCCACUGCUGAGGGUGGGGACCAAGUCUGCGUCAUCCUCUCCUCAGUGCCUCAA> hsa-mir-3692 MI0016093CCAUUCCUGCUGGUCAGGAGGAUACUGGAGCAAUAGA UACCAA> hsa-mir-3714 MI0016135GAAGGCAGCAGUGCUCCCCUGUGACGUGCUCCAUCACCGGGCAGGGAAGACACCGCUGCCACCUC> hsa-mir-3907 MI0016410GGGUUGGAAAGCUGUAGGUGUGGAGGGGCAUGGAUACGGGGGCCAUGAGGGUGGGGUCCAGGCUGGACCAGGCCUGCCCUGAGUCCCCCAGCAGGUGCUCCAGGCUGGCUCACACCCUCUGCCUCUCUCUCUUCCUUCCUGGCCCCAACCC> hsa-mir-3908 MI0016412GCCUGAGCAAUGUAGGUAGACUGUUUCUAAAAAAAUAAAAAGUUAAAAAAAUUUAUGUUAACGUGUAAUGUGUUUACUAAUUUUUUUUUUUUUUUUUGGAGACAGAGUCUCCCUCUGUCGCCAGGC> hsa-mir-3909 MI0016413GGUAUGCUGUUGCGCUGUCCUUCCUCUGGGGAGCAGGCUCCGGGGGACAGGGAAAAGCACACAAGGAACUUGUCCUCUAGGGCCUGCAGUCUCAUGGGAGAGUGACAUGCACCAGGACC> hsa-mir-3910-1 MI0016414CUUUUGCUGUCAGUUUUUCUGUUGCUUGUCUUGGUUUUAUGCCUUUUAUAUCAAGGCACAUAAAAGGCAUAAAACCAAGACAAGCAACAAAAAAAGGAUUGAUCACAGAAG> hsa-mir-3910-2 MI0016431UUUUUUUGUUGCUUGUCUUGGUUUUAUGCCUUUUAUGUGCCUUGAUAUAAAAGGCAUAAAACCAAGACAAGCAACAGAAAAA> hsa- mir-3911 MI0016415GGGUGAGGAUGUGUGUGGAUCCUGGAGGAGGCAGAGAAGACAGUGAGCUUGCCAGUUCUGGUUUCCAACACUUCCUUUCCUGCGCUUCGAUUCCCAGAUCUGCACCC> hsa-mir-3912 MI0016416AGAGAGGA
AUGAACAGUUAAAUUAUAACAUGUCCAUAUUAUGGGUUAGUUGUGGACACAUACUAACGCAUAAUAUGGACAUGUUAUAAUUUAACUGUUCCUUUCU> hsa-mir-3913-1 MI0016417UUGUUUAUAAUAAACUGAAAUAUUUGGGACUGAUCUUGAUGUCUGCCAAAACCUUGGCAGACAUCAAGAUCAGUCCCAAAUAUUUCAGUUUAUUAUAGACAG> hsa-mir-3913-2 MI0016418UGUCUAUAAUAAACUGAAAUAUUUGGGACUGAUCUUGAUGUCUGCCAAGGUUUUGGCAGACAUCAAGAUCAGUCCCAAAUAUUUCAGUUUAUUAUAAACA> hsa-mir-3914-1 MI0016419UGGACUUCAGAUUUAACUUCUCAUUUUCUGGUUCCUUCUAAUGAGUAUGCUUAACUUGGUAGAAGGAACCAGAAAAUGAGAAGUUGAGUAGGAACUCUA> hsa-mir-3914-2 MI0016421GAGUUCCUACUCAACUUCUCAUUUUCUGGUUCCUUCUACCAAGUUAAGCAUACUCAUUAGAAGGAACCAGAAAAUGAGAAGUUAAAUCUGAAGUC> hsa-mir-3915 MI0016420CAAGUUGGCACUGUAGAAUAUUGAGGAAAAGAUGGUCUUAUUGCAAAGAUUUUCAAUAAGACCAUCCUUUCCUCAAUAUUCUGUGGUGUCAUCUUUG> hsa-mir-3916 MI0016422AUCCCAGAGAAGAAGGAAGAAGAGGAAGAAAUGGCUGGUUCUCAGGUGAAUGUGUCUGGGUUCAGGGGAUGUGUCUCCUCUUUCUUCUGGGAU> hsa-mir-3917 MI0016423GGCGCUUUUGUGCGCGCCCGGGUCUUGGUGCUCUGUGCGCGCCGGGUCUUGGUGCUCAG UGGAGACUGCUCAAGGUCAAAGGGGUCUCCAGCUGGGACCCUGCACCUGGUUCGUAGCCCCU> hsa-mir-3919 MI0016425CCUGAGCACCAUUUACUGAGUCCUUUGUUCUCUACUAGUUUGUAGUAGUUCGUAGCAGAGAACAAAGGACUCAGUAAAUGGUGCUCAGG> hsa-mir-3920 MI0016427ACUGAGUGAGGGAGUCAGAGAGUUAAGAGAAUUAGUACAGGUGAGAUUGUACUGAUUAUCUUAACUCUCUGACCCCCUCACUCAGU> hsa-mir-3921 MI0016428CCUAGCCCAGUACAAGGCAUAUGGUACUCAAGAGACUUAGAAAUCCCUAAGUCUCUGAGUACCAUAUGCCUUGUACUGGGCUAGG> hsa-mir-3922 MI0016429GGAAGAGUCAAGUCAAGGCCAGAGGUCCCACAGCAGGGCUGGAAAGCACACCUGUGGGACUUCUGGCCUUGACUUGACUCUUUC> hsa-mir-3923 MI0016430GGUAGAGUGAGCUCUAAUCCAAUAUUACUAGCUUCUUUAUAAGAAGAGGAAACUAGUAAUGUUGGAUUAGGGCUCACUCUACU> hsa-mir-3924 MI0016432UAAAUGAAAAAGUAGUAGUCAAAUAUGCAGAUCUAUGUCAUAUAUACAGAUAUGUAUAUGUGACUGCUACUUUUUUGUUUA> hsa-mir-3925 MI0016433GUGGGAAUAGCAAGAGAACUGAAAGUGGAGCCUGUCACAUCUCCAGACUCCAGUUUUAGUUCUCUUGCUAUUUCCAC > hsa-mir-3926-1 MI0016434AAAAUGGAGCUGGCCAAAAAGCAGGCAGAGACUUUAAAAGCGUCUCUGCCUGCUUUUGGCCAGCUCCGUUUU> hsa-mir-3926-2 MI0016437GGAGCUGGCCAAAAAGCAGGCAGAGACGCUUUUAAAGUCUGCCUGCUU ir-3927 MI0016435UGCCAAUGCCUAUCACAUAUCUGCCUGUCCUAUGACAAACAUGGCAGGUAGAUAUUUGAUAGGCAUUGGCA> hsa-mir-3928 MI0016438GCUGAAGCUCUAAGGUUCCGCCUGCGGGCAGGAAGCGGAGGAACCUUGGAGCUUCGGC> hsa-mir-3929 MI0016439AGUGGCUCACACCAGUAAUCCCAGCACUUUGGGAGGCUGAUGUGAGUAGACCACU> hsa-mir-3934 MI0016590CACAGCCCUUCCUGUCCCCAGUUUUCAGGUGUGGAAACUGAGGCAGGAGGCAGUGAAGUAACUUGCUCAGGUUGCACAGCUGGGAAGUGGAGCAGGGAUUUGAAUCC> hsa-mir-3935 MI0016591GGAUGUGUUCCUGUCCCAGAAGGAGCUGAUGGUUGUAUCUAUGAAGGUAAGCAUUUUUGUAGAUACGAGCACCAGCCACCCUAAGCAAAGGCAGAGAAUGCUUA> hsa-mir-3936 MI0016592AUGAUUCAGAGCAUCUGUCCAGUGUCUGCUGUAGAUCCCUCAAAUCCGUGUUUGGACGCUUCUGGUAAGGGGUGUAUGGCAGAUGCACCCGACAGAUGCACUUGGCAGCA> hsa-mir-3937 MI0016593AGAAGAAUGCCCAACCAGCCCUCAGUUGCUACAGUUCCCUGUUGUUUCAGCUCGACAACAACAGGCGGCUGUAGCAAUGGGGGGCUGGAUGGGCAUCUCAAUGUGC> hsa-mir -3938 MI0016594AGGAAUUUUUAACCCGAUCACUAGAUUAUCUACAAGGGAAUUUUUUUUAAUUUAAAAAAUUCCUUGUAGAUAACCCGGUGGUCAGGUUGGAUGGCUCCAUG> hsa-mir-3939 MI0016596CUGGCUUCCAAAGGCCUCUGUGU CACUGGCUUCCAAACACAUCU> hsa-mir-3940 MI0016597GCUUAUCGAGGAAAAGAUCGAGGUGGGUUGGGGCGGGCUCUGGGGAUUUGGUCUCACAGCCCGGAUCCCAGCCCACUUACCUUGGUUACUCUCCUUCCUUCU> hsa-mir-3941 MI0016598GAGUCAGAAUUCUCAUCAGGCUGUGAUGCUCAGUUGUGUGUAGAUUGAAAGCCCUAAUUUUACACACAACUGAGGAUCAUAGCCUGAUGGUUCCUUUUUGUUU> hsa-mir-3942 MI0016599UCUUCAGUAUGACACCUCAAAGAAGCAAUACUGUUACCUGAAAUAGGCUGCGAAGAUAACAGUAUUUCAGAUAACAGUAUUACAUCUUUGAAGUGUCAUAUUCACUGAC> hsa-mir-3943 MI0016600CACACAGACGGCAGCUGCGGCCUAGCCCCCAGGCUUCACUUGGCGUGGACAACUUGCUAAGUAAAGUGGGGGGUGGGCCACGGCUGGCUCCUACCUGGAC> hsa-mir-3944 MI0016601UCCACCCAGCAGGCGCAGGUCCUGUGCAGCAGGCCAACCGAGAAGCGCCUGCGUCUCCCAUUUUCGGGCUGGCCUGCUGCUCCGGACCUGUGCCUGAUCUUAAUGCUG> hsa-mir-3945 MI0016602GAUGUUGAUGCACGUGACGGGGAGGGCAUAGGAGAGGGUUGAUAUAAAAUGCAAUUACAGCCUCUUAUGCUUUCCAAAGUGGGGGAUGAUUCAAUGAU> hsa-mir-3960 MI0016964GGCGCCCCGGCUCCCCGCGCCCCCGAUCGGGGCCGCCGCUAGUAGUGGCGGCGGCGGAGGCGGGGGCAGCGGCGGCGGCGGCGGAGGCGCC > hsa-mir-3972 MI0016990GCCCAUUUGCCUUGGCUUGGGGUGGCAGUCCUGUGGGAAUGAGAGAUGCCAAACUGGACCUGCCAGCCCCGUUCCAGGGCACAG CAU> hsa-mir-3973 MI0016991GCCCAGGGUAGCUCUCUGUAUUGCUUGUUACAUUUUAGGAUUGCUUGCCCCUUGCUCCAAUGGUGCAGGCAGAAGAAAUGCAAACAAAGUACAGCAUUAGCCUUAGC> hsa-mir-3974 MI0016992GUUCAGGGAAAAGGUCAUUGUAAGGUUAAUGCACCCAUAUUUUAAUAUCAAACUAUGACAAAUUUGACUACAGCCUUUCCGUACCCCUGCCAAAAC> hsa-mir-3975 MI0016993CAUGUAGGUGAGUGAUUGCUAUUUCAAAAGACUAGAGGUAGGAAAUGAGGCUAAUGCACUACUUCACAUG> hsa-mir-3976 MI0016994UGAGGGAUAUAGAGAGCAGGAAGAUUAAUGUCAUAUUGGAGUUGGACUGCAGGGCUUCCUUUACACAAUAAAUAUUGUAUGAAGUGCUGAUGUAACCUUUACUGCAGCAUGACAUGGGAUUUGGCUGUUUUUAUGGCUC> hsa-mir-3977 MI0016995UUGUGCUUCAUCGUAAUUAACCUUAAGUGGUUCGGGUAAAUCACUUUAAUUUGUUAUGUGUUGGCAGAAU> hsa-mir-3978 MI0016996UCAGUGGAAAGCAUGCAUCCAGGGUGUGGAGCCAAAAUUAGAAGGGCCAAAAUUCUACCUGGCCCACUACCACAGCAACCUUGGGCAUCGUUUUCUUUUGA> hsa-mir-4251 MI0015861CACGUCCUCCAGCUUUUUUCCUUAGUGGCCAAUUCCUGAGAAAAGGGCCAACGUGCUUCCA > hsa-mir-4252 MI0015864UGGGGGGCUGGCAGCUCAUCAGUCCAGGCCAUCUGGCCACUGAGUCAGCACCAGCGCCCAAUC> hsa-mir-4253 MI0015860CCAGCCAUCGCCCUUGAGGGGCCCUAGGACUUACUUGCGCAUGUCCAGGGGGUCCAGGUCUGC> hsa I0015862CUUGGGAGGAGGGUGGGGUGGCUCCUCUGCAGUGAGUAGGUCUGCCUGGAGCUACUCCACCAUCUCCCCCAGCCCC> hsa-mir-4255 MI0015863GAGCAUCCUUCAGUGUUCAGAGAUGGAGUCAGUAUUGGUCUGGCCAUUUUUAGGGCAAAGAGGCAGCAUCAU> hsa-mir-4256 MI0015855UGUUCCAUUUAUCUGACCUGAUGAAGGUCUCCUGGCAUUGAUUAGGUCUGAUGAUCCAUUUCUG> hsa-mir-4257 MI0015856GGCUUAGAAACAGUCCCUAGGUAGGAUUUGGGGAGGAGCUAAGAAGCCCCUACAGGGCCCAGAGGUGGGGACUGAGCCUUAGUUGG> hsa-mir-4258 MI0015857ACGCCCCCCGCCCCGCCACCGCCUUGGAGGCUGACCUCUUACUUUCGGUCGGUCUUCUUCCCUGGGCUUGGUUUGGGGGCGGGGGAGUGUC> hsa-mir-4259 MI0015858GAUGGGCCCCUUGUGUCCUGAAUUGGGUGGGGGCUCUGAGUGGGGAAAGUGGGGGCCUAGGGGAGGUCACAGUUGGGUCUAGGGGUCAGGAGGGCCCAGGA> hsa-mir-4260 MI0015859AACAAGGUGACUUGGGGCAUGGAGUCCCACUUCCUGGAGCCCACACCCCAGCUUGUCACACACCAAC> hsa-mir-4261 MI0015868GGUGGAAGUGGGUUCCUCCCAGUUCCUGAGACAGGAAACAGGGACCCAGGAGACCAGC > hsa-mir-4262 MI0015872GAAAGCUGCAGGUGCUGAUGUUGGGGGGACAUUCAGACUACCUGCAGCAGAGCC> hsa-mir-4263 MI0015876AUAGUGCUCUUCAGGGUUUUACUUGGGAGAUUGGAGUGGCCAGUGUUCCUAAACAAUUCUAAGCUUGGCCCAAC ACUCAGUCAUGGUCAUUGUAACAUGAUAGUGACAGGUACUGGGUAAGACUGCAUAG> hsa-mir-4265 MI0015869UGCAGUGGGUUGGAGCUUCAGCCUACACCUGUAAAGAAUUGGUCAGCCUGGGGACUGGUGAUCUCUGCAGCUGUGGGCUCAGCUCUGGGCUGGGCCUGG> hsa-mir-4266 MI0015870CCACUGCUGGCCGGGGCCCCUACUCAAGGCUAGGAGGCCUUGGCCAAGGACAGUC> hsa-mir-4267 MI0015871CUCAGCAGGCUCCAGCUCGGUGGCACUGGGGGAAGGCUCCAGACCCCAGCCUCUGUCAUCCCUGCAUGGAGCCCACAUCUCC> hsa-mir-4268 MI0015874AUGCACAUCAGGUUCUAGAGGUUUUGCCCUAGCGGCUCCUCCUCUCAGGAUGUGAUGUCACCUG> hsa-mir-4269 MI0015875ACAGCGCCCUGCAGGCACAGACAGCCCUGGCUUCUGCCUCUUUCUUUGUGGAAGCCACUCUGUCAGGCCUGGGAUGGAGGGGCA> hsa-mir-4270 MI0015878ACAAAUAGCUUCAGGGAGUCAGGGGAGGGCAGAAAUAGAUGGCCUUCCCCUGCUGGGAAGAAAGUGGGUC> hsa-mir-4271 MI0015879AAAUCUCUCUCCAUAUCUUUCCUGCAGCCCCCAGGUGGGGGGGAAGAAAAGGUGGGGAAUUAGAUUC > hsa-mir-4272 MI0015880UUUUCUGCAAAUUAAUCAGUUAAUGCAUAGAAAGCAUUCAACUAGUGAUUGUGUUAUAAGAG> hsa-mir-4273 MI0015881UCCCCUGUGUGUGUUCUCUGAUGGACAGUAAGCCUUGACUUAUGGCUAAAUGCUUG CUACCUUGUCAGGUGACCCAGCAGUCCCUCCCCCUGCAUGGUGCCC> hsa-mir-4275 MI0015883ACAUUUUUGUCCAAUUACCACUUCUUUUUGCCACCUGAGCACAGUCAGCAGUCAGCAUAAAAAAGUGAUAAUGGGAAGUUAAUGUCU> hsa-mir-4276 MI0015882CACAGUCUGACUCAGUGACUCAUGUGCUGGCAGUGGCCACGUAAAUAGAGCUACUGUGUCUGAAAGCAAU> hsa-mir-4277 MI0015886CUGGGUCGAGGCAGUUCUGAGCACAGUACACUGGGCUGCCCCCACUGCCCAGUGCCCUGCUCAGCUCAAGUCCUUGUGCCCCUC> hsa-mir-4278 MI0015888AUCUAACACCAGGAGAAUCCCAUAGAACAUUGACAUCAACACUAGGGGGUUUGCCCUUGUGGGGAAGAA> hsa-mir-4279 MI0015887UGCUCUGUGGAGCUGAGGAGCAGAUUCUCUCUCUCUCCUCCCGGCUUCACCUCCUGAG> hsa-mir-4280 MI0015889AAUCAGGGUGGAGUGUAGUUCUGAGCAGAGCCUUAAAGGAUGAGGUAUGUUCAAGACUGAAUGACACCUUUGUGAU> hsa-mir-4281 MI0015885GCUGGGGGUCCCCCGACAGUGUGGAGCUGGGGCCGGGUCCCGGGGAGGGGGGUUCUGGGCAG > hsa-mir-4282 MI0015890GGUGAAGUUCCAGGGGAAGAUUUUAGUAUGCCACAUUUCUAAAAUUUGCAUCCAGGAACAUCAUCCU> hsa-mir-4283-1 MI0015892ACUCUGAUCCUGGGGCUCAGCGAGUUUGCAAGGGGUGUUUCUGUCCAUGGUCAGGCUUGCCAGCCUUGGUCCUUGGGCCC> hsa-mir-4283-2 MI0015982ACUCUGAUCCUGGGGCUCAGCGAGUUUGCAAGGGGUGUUUCUGUCCAUGGUCAGGCUUGCCAGCCUU GGUCCUUGGGCCC> hsa-mir-4284 MI0015893GUUCUGUGAGGGGCUCACAUCACCCCAUCAAAGUGGGGACUCAUGGGGAGAGGGGGUAGUUAGGAGCUUUGAUAGAGGCGG> hsa-mir-4285 MI0015891AUUAGCUGGGGCGGCGAGUCCGACUCAUCAAUAUUUUAAGGAAUGACCCGGCCUUGGGGUGCGGAAUUGCUGCGCGGGCGGGGGC> hsa-mir-4286 MI0015894UACUUAUGGCACCCCACUCCUGGUACCAUAGUCAUAAGUUAGGAGAUGUUAGAGCUGUGAGUACCAUGACUUAAGUGUGGUGGCUUAAACAUG> hsa-mir-4287 MI0015895UAGUUCUUUUUCUCCCUUGAGGGCACUUUUCAGUUCCUGAGAUCAAUGUGGUCCCUACUGGGGAGACCAUAGGAGCCC> hsa-mir-4288 MI0015896AUGGAGGUGGAGAGUCAUCAGCAGCACUGAGCAGGCAGUGUUGUCUGCUGAGUUUCCACGUCAUUUG> hsa-mir-4289 MI0015898CCUUGGGAGGGCAUUGUGCAGGGCUAUCAGGCAGUUUCCUGGGCCCUGUCUGCAGAGCCUAAACAGAUCA> hsa-mir-4290 MI0015899GCCACCAAGAAGGUGAAGGGAGGGUCAGUCCCAAUCUGAAUCCCACCAAAAUAGGUGGUAGAGGGUUGCCCUCCUUUCUUCCCUCACCUCUGACC > hsa-mir-4291 MI0015900CGCCGGGGGCUUCAGCAGGAACAGCUGGGUGGAGGCAGAGCUGUUCUGCUGUGGCUGCAGCCCUG> hsa-mir-4292 MI0015897GAGACACCAGAAGGCCACCUGCUUAGGAGGCCAGAGGUGCCCGGGCCGGCCUUGGUGAGGCGCCUAG UGACAGGAACAGCCUGACUGAA> hsa-mir-4294 MI0015827CCGAUGCCUCGGGAGUCUACAGCAGGGCCAUGUCUGUGAGGGCCCAAGGGUGCAUGUGUCUCCCAGGUUUCGGUGC> hsa-mir-4295 MI0015822CUUUGUGGAACAGUGCAAUGUUUUCCUUGCCUGUGGCAAGACCACUUCGGUUCAAGGCUAAGAAACUAGACUGUUCCUACAGAGA> hsa-mir-4296 MI0015823UUGGGCUUUGAUGUGGGCUCAGGCUCAGAGGGCUGAAGUGGUUGUGGGGAGGGGCUUCUGGGGACUGUGUCCAUGUCUCUGUCGUUUU> hsa-mir-4297 MI0015824AGCACGCACGUGCCUUCCUGUCUGUGCCUGCCUUCGAAGUGCACGGCAGGGCCAGGACGGGUCGCUGUGGGUGGGG> hsa-mir-4298 MI0015830GGGGAGGUACCUGGGACAGGAGGAGGAGGCAGCCUUGCCUCAGAAACCAAACUGUCAAAAGUGUAGGUUCCAC> hsa-mir-4299 MI0015829GGGUUCUGACCAAUCAUGUUACAGUGUUUUCUCCUUUAGAGAGAGCUGGUGACAUGAGAGGCAGAAAAAGGA> hsa-mir-4300 MI0015831UGAGUUUAGAAGAGGGCCAGCUAAAUCAGCAGAGACAUGAGGUGAUCAAAAACCUUUUUUCAAAGCAGUGGGAGCUGGACUACUUCUGAACCAAUA > hsa-mir-4301 MI0015828ACCAGCCACCUCCCACUACUUCACUUGUGAACAUUGCAUUCGUGGAGGGUGGCAGGUGCAGCUCUG> hsa-mir-4302 MI0015833UCAGGAGGGACCAGUGUGGCUCAGCGAGGUGGCUGAGUUUACUUAAGGUAUUGGAAUGAG
AGCUGAGGACAGCUUGCUCUGCUUUUCUUUAGCUUAGGAGCUAACCAUGGU> hsa-mir-4304 MI0015832AGAGAAGUGGCCGGCAUGUCCAGGGCAUCCCCAUUGCUCUGUGACUGCUGCCAUCCUUCUCC> hsa-mir-4305 MI0015835CUGCCUUAGACCUAGACACCUCCAGUUCUGGGUUCUUAGAGGCCUAAUCCUCUACAAACUCAGUUUUCAGACUGUGAGGGAAAUUCUCUGUCUUAUUGCUUU> hsa-mir-4306 MI0015836AAGCUGCUUAGUGUCCUUAGAGUCUCCAGAGGCAUCCCUAACCCAGAAUCUUUUGACUGUCCUCUGGAGAGAAAGGCAGUAGGUCUGUACC> hsa-mir-4307 MI0015838UCAGAAGAAAAAACAGGAGAUAAAGUUUGUGAUAAUGUUUGUCUAUAUAGUUAUGAAUGUUUUUUCCUGUUUCCUUCAGGGCCA> hsa-mir-4308 MI0015839UAUGGGUUCAGAGGGAACUCCAUUGGACAGAAAUUUCCUUUUGAGGAAAUCUUUCCCUGGAGUUUCUUCUUACCUUUUUCC> hsa-mir-4309 MI0015837UCUGGGGGUUCUGGAGUCUAGGAUUCCAGGAUCUGGGUUUUGAGGUCUUGGGUUGUAGGGUCUGCGGUUUGAAGCCCCUCUUG> hsa-mir-4310 MI0015840UGGCGUCUGGGGCCUGAGGCUGCAGAACAUUGCAGCAUUCAUGUCCCACCCCCACCA > hsa-mir-4311 MI0015841UCAGAGAGGAAAGAGAGCUGAGUGUGACCUGGAGCAGCUCAGGAGGGCUUCCUGGGUGAGGUGGCAGGUUACAGGUUCGAUCUUGGCCCUCAGAUUC> hsa-mir-4312 MI0015842GAAAGGUUGGGGGCACAGAGCAAG ir-4313 MI0015843GAUCAGGCCCAGCCCCCUGGCCCCAAACCCUGCAGCCCCAGCUGGAGGAUGAGGAGAUGCUGGGCUUGGGUGGGGGAAUCAGGGGUGUAAAGGGGCCUGCU> hsa-mir-4314 MI0015846GGCCAUUCCUCUCUGGGAAAUGGGACAGGUAGUGGCCACAGUGAGAAAGCUGGCCUGUCCUUCUGCCCCAGGGCCCAGAGUCUGUGACUGGA> hsa-mir-4315-1 MI0015844UGGGCUUUGCCCGCUUUCUGAGCUGGACCCUCUCUCUACCUCUGGUGCAGAACUACAGCGGAAGGAAUCUCUG> hsa-mir-4315-2 MI0015983UGGGCUUUGCCCGCUUUCUGAGCUGGACCCUCUCUCUACCUCUGGUGCAGAACUACAGCGGAAGGAAUCUCUG> hsa-mir-4316 MI0015845AGUGGCCCAGGGUGAGGCUAGCUGGUGUGGUCACCCACUCUCCAGCCCAGCCCCAAUCCCACCACAACCAC> hsa-mir-4317 MI0015850AAAAGGCGAGACAUUGCCAGGGAGUUUAUUUUGUAGCUCUCUUGAUAAAAUGUUUUAGCAAACAC> hsa-mir-4318 MI0015847GCUUCUUAAUUAUGUCAUAAACCCACUGUGGACAAGGGCCUUGUCUUAGACAGUCACUGUGGGUACAUGCUAGGUGCUCAA > hsa-mir-4319 MI0015848UUGGCUUGAGUCCCUGAGCAAAGCCACUGGGAAUGCUCCCUGAGGACGUUAUAUGAGUGCUCAGCUCAUGGGGCUAUGAUGGUCA> hsa-mir-4320 MI0015849GACAUGUGGGGUUUGCUGUAGACAUUUCAGAUAACUCG UGCGGGGGCCUGGC> hsa-mir-4322 MI0015851ACCGCGAGUUCCGCGCCUGGCCGUGUCGCCCCACGAGGGGGACUGUGGGCUCAGCGCGUGGGGCCCGGAGCAU> hsa-mir-4323 MI0015853CGGGGCCCAGGCGGGCAUGUGGGGUGUCUGGAGACGCCAGGCAGCCCCACAGCCUCAGACCUCGGGCAC> hsa-mir-4324 MI0015854CGGCCCCUUUGUUAAGGGUCUCAGCUCCAGGGAACUUUAAAACCCUGAGACCCUAACCUUAAAGGUGCUGCA> hsa-mir-4325 MI0015865GGGGAAGAUGUUGCACUUGUCUCAGUGAGAGAUGCUUCUAGAUCCAGGAGGCAGACCUCAAGGAUGGAGAGAAGGCAGAUCCUUUGAGAU> hsa-mir-4326 MI0015866GCUGCUCUGCUGUUCCUCUGUCUCCCAGACUCUGGGUGGAUGGAGCAGGUCGGGGGCCA> hsa-mir-4327 MI0015867GGCCUGGGUAGGCUUGCAUGGGGGACUGGGAAGAGACCAUGAACAGGUUAGUCCAGGGAGUUCUCAUCAAGCCUUUACUCAGUAG> hsa-mir-4328 MI0015904AACAGUUGAGUCCUGAGAACCAUUGAGAACCAGUUUUCCCAGGAUUAACUGUUCCG > hsa-mir-4329 MI0015901UAGAGAGGAAGGUGUACCAGGGUUUUGGAGUUUUUUUUUCCUCCUGAGACCCUAGUUCCACAUUCUGGAGC> hsa-mir-4330 MI0015902AAUUGUCAGCAGGCAAUUAUCUGAGGAUGCAGGAGAGGAAGGGGGCUUCUUUUUGACGCCUACUUCAUCAGCUGCUCCUCAGAUCAGAGCCUUGCAGGUCAGGCC> hsa-mir-4417 MI0016753GAAAACAACCAGGUGGGCUUCCCGGAGGGCGGAACACCCAGCCCCAGCAUCCAGGGCUCACCUACCACGUUUG> hs a-mir-4418 MI0016754UGGUUUUUGCUCUGAGUGACCGUGGUGGUUGUGGGAGUCACUGCAGGACUCAGCAGGAAUUC> hsa-mir-4419a MI0016755UGGUGGUGUGUGCCUGUAGUCUUAGCUACUCGGGAGGCUGAGGGAGGAGACUGCAGUGAGUGGAGGUCACGCCACUG> hsa-mir-4419b MI0016861CUCAGGCUCAGUGGUGCAUGCUUAUAGUCCCAGCCACUCUGGAGGCUGAAGGAAGAUGGCUUGAGCCU> hsa-mir-4420 MI0016757CUCUUGGUAUGAACAUCUGUGUGUUCAUGUCUCUCUGUGCACAGGGGACGAGAGUCACUGAUGUCUGUAGCUGAGAC> hsa-mir-4421 MI0016758CUGGGUCUCCUUUCUGCUGAGAGUUGAACACUUGUUGGGACAACCUGUCUGUGGAAAGGAGCUACCUAC> hsa-mir-4422 MI0016759AGUUCUUCUGCAGACAAAAGCAUCAGGAAGUACCCACCAUGUACCAGUGGGCCCUUCUUGAUGCUCUUGAUUGCAGAGGAGCC> hsa-mir-4423 MI0016760AUCAUGUACUGCAGUUGCCUUUUUGUUCCCAUGCUGUUUAAGCCUAGCAUAGGCACCAAAAAGCAACAACAGUAUGUGAA> hsa -mir-4424 MI0016763CUUACAUCACACACAGAGUUAACUCAAAAUGGACUAAUUUUUCCACUAGUUAGUCCAUUUCAAGUUAACUCUGUGUGUGAUGUAGU> hsa-mir-4425 MI0016764GUGCUUUACAUGAAUGGUCCCAUUGAAUCCCAACAGCUUUGCGAAGUGUUGUUGGGAUUCAGCAGGACCAUUCGUGUAAAGUAA> hsa-mir-4426 MI0016765AGUUGGAAGAUGGACGUACUUUGUCUGACUACAAUAUUCAAAAGGAGUCUACUCUUCAUCUUG> hsa-mir-4427 MI001676 6GAAGCCUCUUGGGGCUUAUUUAGACAAUGGUUUCAUCAUUUCGUCUGAAUAGAGUCUGAAGAGUCUUU> hsa-mir-4428 MI0016767UUGGCAGGUGCCAUGUUGCCUGCUCCUUACUGUACACGUGGCUGGCAAGGAGACGGGAACAUGGAGCCGCCAU> hsa-mir-4429 MI0016768AGGGAGAAAAGCUGGGCUGAGAGGCGACUGGUGUCUAAUUUGUUUGUCUCUCCAACUCAGACUGCCUGGCCCA> hsa-mir-4430 MI0016769GUGAGGCUGGAGUGAGCGGAGAUCGUACCACUGCACUCCAACCUGGUGA> hsa-mir-4431 MI0016771UGGUUUGCGACUCUGAAAACUAGAAGGUUUAUGACUGGGCAUUUCUCACCCAAUGCCCAAUAUUGAACUUUCUAGUUGUCAGAGUCAUUAACCC> hsa-mir-4432 MI0016772GCAUCUUGCAGAGCCGUUCCAAUGCGACACCUCUAGAGUGUCAUCCCCUAGAAUGUCACCUUGGAAAGACUCUGCAAGAUGCCU> hsa-mir-4433 MI0016773CAUCCUCCUUACGUCCCACCCCCCACUCCUGUUUCUGGUGAAAUAUUCAAACAGGAGUGGGGGUGGGACAUAAGGAGGAUA> hsa-mir-4434 MI0016774UCACUUUAGGAGAAGUAAAGUAGAACUUUGGUUUUCAACUUUUCCUACAGUGU > hsa-mir-4435-1 MI0016775AGGCAGCAAAUGGCCAGAGCUCACAGAGGGAUGAGUGCACUUCACCUGCAGUGUGACUCAGCAGGCCAACAGAUGCUA> hsa-mir-4435-2 MI0016777GCAAAUGGCCAGAGCUCACAGAGGGAUGAGUCCACUUCACCUGCA GAAUCUGCUCCACGAUUUGGGCAGGACAGGCAGAAGUGGAUAAGUGAGGA> hsa-mir-4436b-1 MI0017425GUGUCCUCACUUGUCCACUUCUGCCUGCCCUGCCCAAAUGGUGGAGCAGAUUCGAGGGGCAGGGCAGGAAGAAGUGGACAAGUGAGGCCAU> hsa-mir-4436b-2 MI0019110GUGUCCUCACUUGUCCACUUCUGCCUGCCCUGCCCAAAUGGUGGAGCAGAUUCGAGGGGCAGGGCAGGAAGAAGUGGACAAGUGAGGCCAU> hsa-mir-4437 MI0016778ACUUUGUGCAUUGGGUCCACAAGGAGGGGAUGACCCUUGUGGGCUCAGGGUACAAAGGUU> hsa-mir-4438 MI0016781UAAGUGUAAACUUAAGGACUGUCUUUUCUAAGCCUGUGCCUUGCCUUUCCUUUGGCACAGGCUUAGAAAAGACAGUCUUUAAGUUUACACUUC> hsa-mir-4439 MI0016782CCAGUGACUGAUACCUUGGAGGCAUUUUAUCUAAGAUACACACAAAGCAAAUGCCUCUAAGGUAUCAGUUUACCAGGCCA> hsa-mir-4440 MI0016783CUCUCACCAAGCAAGUGCAGUGGGGCUUGCUGGCUUGCACCGUGACUCCCUCUCACCAAGCAAGUGUCGUGGGGCUUGCUGGCUUGCACUGUGAAGAU> hsa- mir-4441 MI0016784CAGAGUCUCCUUCGUGUACAGGGAGGACUGUACGUGAGAGAUAGUCAGAUCCGCAUGUUAGAGCAGAGUCUCCUUCGUGUACAGGGAGGAGAUUGUAC> hsa-mir-4442 MI0016785GCGCCCUCCCUCUCCCCGGUGUGCAAAUGUGUGUG -mir-4444-1 MI0016787GUGACGACUGGCCCCGCCUCUUCCUCUCGGUCCCAUAUUGAACUCGAGUUGGAAGAGGCGAGUCCGGUCUCAAA> hsa-mir-4444-2 MI0019111GUGACGACUGGCCCCGCCUCUUCCUCUCGGUCCCAUAUUGAACUCGAGUUGGAAGAGGCGAGUCCGGUCUCAAA> hsa-mir-4445 MI0016788UUCCUGCAGAUUGUUUCUUUUGCCGUGCAAGUUUAAGUUUUUGCACGGCAAAAGAAACAAUCCAGAGGGU> hsa-mir-4446 MI0016789CUGGUCCAUUUCCCUGCCAUUCCCUUGGCUUCAAUUUACUCCCAGGGCUGGCAGUGACAUGGGUCAA> hsa-mir-4447 MI0016790GUUCUAGAGCAUGGUUUCUCAUCAUUUGCACUACUGAUACUUGGGGUCAGAUAAUUGUUUGUGGUGGGGGCUGUUGUUUGCAUUGUAGGAU> hsa-mir-4448 MI0016791AGGAGUGACCAAAAGACAAGAGUGCGAGCCUUCUAUUAUGCCCAGACAGGGCCACCAGAGGGCUCCUUGGUCUAGGGGUAAUGCCA> hsa-mir-4449 MI0016792AGCAGCCCUCGGCGGCCCGGGGGGCGGGCGGCGGUGCCCGUCCCGGGGCUGCGCGAGGCACAGGCG> hsa-mir-4450 MI0016795UGUCUGGGGAUUUGGAGAAGUGGUGAGCGCAGGUCUUUGGCACCAUCUCCCCUGGUCCCUUGGCU> hsa-mir-4451 MI0016797UCUGUACCUCAGCUUUGCUCCCAACCAACCACUUCCACAUGUUUUGCUGGUAGAGCUGAGGACAGC> hsa-mir-4452 MI0016798UGGAUCACUUGAGGCCAAGAGUGCAAGGCUGUAGUGUGCACAGCCUUGAAUUCUUGGCCUUAAGUGAUCCC> hsa-mir-4453 MI0016799UGGAGAGCUUGGUCUGU AGCGGUUUCCUUCGGGGCAGGUGGGGACUGCUCCUUUGGGAGGAAGGAGGAGGCCCAGGCCGCGUCUUCAGG> hsa-mir-4454 MI0016800CCGGAUCCGAGUCACGGCACCAAAUUUCAUGCGUGUCCGUGUGAAGAGACCACCA> hsa-mir-4455 MI0016801AGAAGGGUGUGUGUGUUUUUCCUGAGAAUAAGAGAAGGAAGGACAGCCAAAUUCUUCA> hsa-mir-4456 MI0016802AUGAACCUGGUGGCUUCCUUUUCUGGGAGGAAGUUAGGGUUCA> hsa-mir-4457 MI0016803GGAGUACUCCAGUCAAUACCGUGUGAGUUAGAAAAGCUCAAUUCACAAGGUAUUGACUGGCGUAUUCA> hsa-mir-4458 MI0016804GAGCGCACAGAGGUAGGUGUGGAAGAAAGUGAAACACUAUUUUAGGUUUUAGUUACACUCUGCUGUGGUGUGCUG> hsa-mir-4459 MI0016805ACCCAGGAGGCGGAGGAGGUGGAGGUUGCAGUGAGCCAAGAUCGUGGCACUGACUCCAGCCUGGGG> hsa-mir-4460 MI0016806GUUUUUUGCCCAUAGUGGUUGUGAAUUUACCUUCUCCUCUUUGCAGUGAUAAAGGAGGUAAAUUCACAACCACUGUGGGCAGAAAC > hsa-mir-4461 MI0016807GAGUAGGCUUAGGUUAUGUACGUAGUCUAGGCCAUACGUGUUGGAGAUUGAGACUAGUAGGGCUAGGCCUACUG> hsa-mir-4462 MI0016810CUUCCCAGCUGCCCUAAGUCAGGAGUGGCUUUCCUGACACGGAGGGUGGCUUGGGAAA> hsa-mir-4463 MI0016811AAUAGAUUAUUGGUCACCACCUCCAGUUUCUGAAUUUGUGAGACUGGGGUGGGGCCUGAGAAUUUGC> hsa-mir-4464 MI0016812GGAACCUUAGUAAGGUUUGGAUAGA UGCAAUAAAGUAUGUCCACAGCUGAAAGGACAUACUUUAUUGCAUGUAUCCAAACCUUACUAAUUCA> hsa-mir-4465 MI0016816CAUGUGUCCCCUGGCACGCUAUUUGAGGUUUACUAUGGAACCUCAAGUAGUCUGACCAGGGGACACAUGA> hsa-mir-4466 MI0016817ACGCGGGUGCGGGCCGGCGGGGUAGAAGCCACCCGGCCCGGCCCGGCCCGGCGA> hsa-mir-4467 MI0016818UGGUGGCGGCGGUAGUUAUGGGCUUCUCUUUCUCACCAGCAGCCCCUGGGCCGCCGCCUCCCU> hsa-mir-4468 MI0016819AGUCUUCUCCUGGGGCUUUGGUGGCUAUGGUUGACUGGGCCACUCAGAGCAGAAGGAUGAGAUG> hsa-mir-4469 MI0016820CCGACGCGGAGAGCGGCUCUAGGUGGGUUUGGCGGCGGCGAGGACACCGCCGCUCCCUCUAGGGUCGCUCGGAGCGUGA> hsa-mir-4470 MI0016821CGAGCCUCUUUCGGCUUUCCAGUUUGUCUCGGUCCUUUGGAACGUGGCAAACGUGGAAGCCGAGAGGGCUCU> hsa-mir-4471 MI0016822CCAAAUUUAAAACUUAAACCUCUACUAAGUUUCCAUGAAAAGAACCCAUGGGAACUUAGUAGAGGUUUAAGUUUUAAAUUUGA > hsa-mir-4472-1 MI0016823UGGCAGACCCUUGCUCUCUCACUCUCCCUAAUGGGGCUGAAGACAGCUCAGGGGCAGGGUGGGGGGUGUUGUUUUUGUUU> hsa-mir-4472-2 MI0016824UGGUGGGGGUGGGGGGUGUUGUUUUUGUUUUUGAGACAGAGUCUUGCUCCGUCGCCCAGGCCGGAGU> hsa-mir-4473 MI0016825AAGGAACAGGGGACACUUGUAAUGGAGAACACUAAGCUAUGGACUGCUAUGGACUGCUAGUGCUCUC CGUUACAAGUAUCCCCUGUUACCU> hsa-mir-4474 MI0016826UUGCCUACCUUGUUAGUCUCAUGAUCAGACACAAAUAUGGCUCUUUGUGGCUGGUCAUGAGGCUAACAAGGUAGGCAC> hsa-mir-4475 MI0016827AUCUCAAUGAGUGUGUGGUUCUAAAUGACUCAUAGUCAAGGGACCAAGCAUUCAUUAUGAA> hsa-mir-4476 MI0016828AAAAGCCUGUCCCUAAGUCCCUCCCAGCCUUCCAGAGUUGGUGCCAGGAAGGAUUUAGGGACAGGCUUUG> hsa-mir-4477a MI0016829UCCUCCUCCCAUCAAUCACAAAUGUCCUUAAUGGCAUUUAAGGAUUGCUAUUAAGGACAUUUGUGAUUCACGGGAGGAGGU> hsa-mir-4477b MI0016830ACCUCCUCCCGUGAAUCACAAAUGUCCUUAAUAGCAAUCCUUAAAUGCCAUUAAGGACAUUUGUGAUUGAUGGGAGGAGGA> hsa-mir-4478 MI0016831GGCCGAGGCUGAGCUGAGGAGCCUCCAAACCUGUAGACAGGGUCAUGCAGUACUAGGGGCGAGCCUCAUCCCCUGCAGCCCUGGCC> hsa-mir-4479 MI0016838GAAACCAAGUCCGAGCGUGGCUGGCGCGGGAAAGUUCGGGAACGCGCGCGGCCGUGCUCGGAGCAGCGCCA > hsa-mir-4480 MI0016841GCAGAGGUGAGUUGACCUCCACAGGGCCACCCAGGGAGUAAGUAGCCAAGUGGAAGUUACUUUACCUCUGU> hsa-mir-4481 MI0016842GGAGUGGGCUGGUGGUUUUUAAGAGGAAGGGAGACCUAAGCUAGCAUGAGCACGCU.
UGGAAAUGUGUGGAAGAUGGCAUUUCUAUUUCUCAGUGGGGCUCUUACC> hsa-mir-4482-2 MI0017872AAUAAAAAUUGAAUAGUGAGCAACCCAGUGGGCUAUGGAAAUGUGUGGAAGAUGGCAUUUCUAUUUCUCAGUGGGGCUCUUACCUAUUACUCAUCAAC> hsa-mir-4483 MI0016844AAAAAACAACAUACUUAGUGCAUACCCAUAUAAUAUUAGGGGUGGUCUGUUGUUGUUUUUCU> hsa-mir-4484 MI0016845GGGUUUCCUCUGCCUUUUUUUCCAAUGAAAAUAACGAAACCUGUUAUUUCCCAUUGAGGGGGAAAAAGGCGGGAGAAGCCCCA> hsa-mir-4485 MI0016846AGAGGCACCGCCUGCCCAGUGACAUGCGUUUAACGGCCGCGGUACCCUAACUGUGCA> hsa-mir-4486 MI0016847GCAUGCUGGGCGAGGCUGGCAUCUAGCACAGGCGGUAGAUGCUUGCUCUUGCCAUUGCAAUGA> hsa-mir-4487 MI0016848ACUGUCCUUCAGCCAGAGCUGGCUGAAGGGCAGAAGGGAACUGUCCUUCAGCCAGAGCUGGCUGAAGGGCAGA> hsa-mir- 4488 MI0016849GGUAGGGGGCGGGCUCCGGCGCUGGGACCCCACUAGGGUGGCGCCUUGGCCCCGCCCCGCCC> hsa-mir-4489 MI0016850GGGGGUGGGGCUAGUGAUGCAGGACGCUGGGGACUGGAGAAGUCCUGCCUGACCCUGUCCCA> hsa-mir-4490 MI0016852AUAGUUUCUGCAAUGCUCAAAUCUCUGGCCAAAGACCAGAACUUAAUGGUCUCUGGUAAGAGAUUUGGGCAUAUUAGAAACUAA> hsa-mir-4491 MI0016853ACAUUUGGUCACACCAGUCCACAUUAACGUGGACCAGACAAUAUUAAUGUGGACUGGUGUGACCAAAA> hsa-mi r-4492 MI0016854CUGCAGCGUGCUUCUCCAGGCCCCGCGCGCGGACAGACACACGGACAAGUCCCGCCAGGGGCUGGGCGCGCGCCAGCCGG> hsa-mir-4493 MI0016855CCAGAGAUGGGAAGGCCUUCCGGUGAUUAUCACAGCCAUGCCUUUACCUCCAGAAGGCCUUUCCAUCUCUGUC> hsa-mir-4494 MI0016856AGUUUUAGUUACCCUGGUCAUCUGCAGUCUGAAAAUACAAAAUGGAAAAUUCCAGACUGUGGCUGACCAGAGGUAACUGAAACC> hsa-mir-4495 MI0016857AAGAAAUGUAAACAGGCUUUUUGCUCAGUGGAGUUAUUUUGAGCAAAAAGCUUAUUUACAUUUCUG> hsa-mir-4496 MI0016858ACAUCAGCUCAUAUAAUCCUCGAAGCUGCCUUUAGAAAUGAGGAAACUGAAGCUGAGAGGG> hsa-mir-4497 MI0016859ACCUCCGGGACGGCUGGGCGCCGGCGGCCGGGAGAUCCGCGCUUCCUGAAUCCCGGCCGGCCCGCCCGGCGCCCGUCCGCCCGCGGGUC> hsa-mir-4498 MI0016860AGGGCUGGGCUGGCAGGGCAAGUGCUGCAGAUCUUUGUCUAAGCAGCCCCUGCCUUGGAUCUCCCA> hsa-mir -4499 MI0016862AAGACUGAGAGGAGGGAACUGGUGAGUUGUACAUAGAAAUGCUUUCUAACUCCUUGUCUCAGUCUGUUU> hsa-mir-4500 MI0016863CAGGAGAGAAAGUACUGCCCAGAAGCUAAAGUGUAGAUCAAACGCAUAAUGGCUGAGGUAGUAGUUUCUUGAACUU> hsa-mir-4501 MI0016864UAUGUGACCUCGGAUGAAUCACUGAAAUAUGUCUGAGCUUCUGUUUCAUCAGAUGUCACAUUUU> hsa-mir-4502 MI0016865AGCCUUUAGCAAGUUGUAAUCUUUUU GCUGAUGGAGGGUCUUGCCUCCAUGGGGAUGGCUGAUGAUGAUGGUGCUGAAGGC> hsa-mir-4503 MI0016866ACAAUGUAGAUAUUUAAGCAGGAAAUAGAAUUUACAUAUAAAUUUCUAUUUGUUUCUAUUUCCUGCUUAAAUAUCUACAUUGC> hsa-mir-4504 MI0016867CUAAGAUAAUGUCCUCCAGGUUCAUCUCUGUUGUCAUUUGUGGCAUGGACCAUUUGUGACAAUAGAGAUGAACAUGGAGGAUAUUAUCUUAA> hsa-mir-4505 MI0016868GGAGGCUGGGCUGGGACGGACACCCGGCCUCCACUUUCUGUGGCAGGUACCUCCUCCAUGUCGGCCCGCCUUG> hsa-mir-4506 MI0016869UGGCCUCUGCCAUCAGACCAUCUGGGUUCAAGUUUGGCUCCAUCUUUAUGAAAUGGGUGGUCUGAGGCAAGUGGUCU> hsa-mir-4507 MI0016871UCUGGGCUGAGCCGAGCUGGGUUAAGCCGAGCUGGGUUGGGCUGGGCUGGGU> hsa-mir-4508 MI0016872AGGACCCAGCGGGGCUGGGCGCGCGGAGCAGCGCUGGGUGCAGCGCCUGCGCCGGCAGCUGCAAGGGCCG> hsa-mir-4509- 1 MI0016873CUUUAAUACUAUCUCAAACUAAAGGAUAUAGAAGGUUUUCCCUUUCUCUUGCCCUGAAACCUUCUGUAUCCUUUAUUUUGAGAUAGUAUUAGAA> hsa-mir-4509-2 MI0016874CUUUAAUACUAUCUCAAACUAAAGGAUAUAGAAGGUUUUCCCUUUCUCUUGCCCUGAAACCUUCUGUAUCCUUUAUUUUGAGAUAGUAUUAGAA> hsa-mir-4509-3 MI0016875CUUUAAUACUAUCUCAAACUAAAGGAUAUAGAAGGUUUUCCCUUUCUCUUGCCCUGAAACCUUCUGUAUCCUUUAUUUUGAGAUAGUAUUAGAA> hs a-mir-4510 MI0016876GUGUAUGUGAGGGAGUAGGAUGUAUGGUUGUUAGAUAGACAACUACAAUCUUUUCUCACAACAGACAG> hsa-mir-4511 MI0016877AAAAAAAAGGGAAAGAAGAACUGUUGCAUUUGCCCUGCACUCAGUUUGCACAGGGUAAAUGCAAUAGUUCUUCUUUCCCUUUUUUUA> hsa-mir-4512 MI0016878CUCAGCCCGGGCAAUAUAGUGAGACCUCGUCUCUACAAAAAAUUGAGACAGGGCCUCACUGUAUCGCCCAGGCUGGA> hsa-mir-4513 MI0016879AUUCUAGGUGGGGAGACUGACGGCUGGAGGCCCAUAAGCUGUCUAAAACUUCGGCCCCCAGAUUUCUGGUCUCCCCACUUCAGAAC> hsa-mir-4514 MI0016880GUUGAGACAGGCAGGAUUGGGGAAACAUCUUUUACCUCGUCUCUUGCCUGUUUUAGA> hsa-mir-4515 MI0016881GCGGGAGGUGUAACAGGACUGGACUCCCGGCAGCCCCAGGGCAGGGGCGUGGGGAGCUGGUCCUAGCUCAGCGCUCCCGGA> hsa-mir-4516 MI0016882AGGGAGAAGGGUCGGGGCAGGGAGGGCAGGGCAGGCUCUGGGGUGGGGGGUCUGUGAGUCAGCCACGGCUCUGCCCACGUCUCCCC> hsa -mir-4517 MI0016883AGGUAAAUAUGAUGAAACUCACAGCUGAGGAGCUUAGCAAGUAGCUAAGGCCAGAGCUUGUGUUUGGGUGGUGUGGCUG> hsa-mir-4518 MI0016884UGGGGGAAAAGUGCUGGGAUUGAUUAGUGAUGUCUGCUGGGGAACCGG 20a MI0016886GUGUGCCACCUGCGUGUUUUCUGUCCAAAUCAGAAAAGGAUUUGGACAGAAAACACGCAGGAAGAAGGAA> hsa-mir-4520b MI0017358CCUGCGUGUUUUCUGUCCAAAUCCUUUUCUGAUUUGGACAGAAAACACGCAGGU> hsa-mir-4521 MI0016887UCGGCUAAGGAAGUCCUGUGCUCAGUUUUGUAGCAUCAAAACUAGGAUUUCUCUUGUUAC> hsa-mir-4522 MI0016889GCGGGCGUUGCCUGGGGGCCUCGCAGGGGGAGAUCCAGCCCAGGCUGGUUCCGCUGACUCUGCCUGUAGGCCGGUGGCGUCUUCUGG> hsa-mir-4523 MI0016890GCGGGGGACCGAGAGGGCCUCGGCUGUGUGAGGACUAGAGGCGGCCGAGGCCCGGGCCGGUUCCCCCGA> hsa-mir-4524a MI0016891GAACGAUAGCAGCAUGAACCUGUCUCACUGCAGAAUUAUUUUGAGACAGGCUUAUGCUGCUAUCCUUCA> hsa-mir-4524b MI0019114UAGCUGGGUGGAUGUGUUCUUUUGAAGGAUAGCAGCAUAAGCCUGUCUCAAAAUAAUUCUGCAGUGAGACAGGUUCAUGCUGCUAUCGUUCCAAAGAGGAAGGGUAAUCACUGUC> hsa-mir-4525 MI0016892GUCAGAGGGGGGAUGUGCAUGCUGGUUGGGGUGGGCUGCCUGUGGACCAAUCAGCGUGCACUUCCCCACCCUGAA> hsa-mir-4526 MI0016893UGCGGUGACAUCAGGGCCCAGUCCCUGCUGUCAUGCCCCAGGUGACGUGCUGGGCUGACAGCAGGGCUGGCCGCUAACGUCACUGUC> hsa-mir-4527 MI0016894CCAGAAGUGGUCUGCAAAGAGAUGACUGUGAAUCCAAGAUCCACAUCAGCUCUGUGCUGCCUACAUCUGA> hsa-mir-4528 MI00168 95UAUUCUACUGAGAGUACAGAUCUUUAUAUAUAUGAUCAUUAUAUGUAUGAUGAGAUCAUUAUAUGUAUGAUCUGGACACCCAGUAGAAUC> hsa-mir-4529 MI0016896AUGACAGGCCAUCAGCAGUCCAAUGAAGACAUGAAGACCCAAUGUCUUCAUUGGACUGCUGAUGGCCCGUCACUGGGA> hsa-mir-4530 MI0016897CGACCGCACCCGCCCGAAGCUGGGUCAAGGAGCCCAGCAGGACGGGAGCGCGGCGC> hsa-mir-4531 MI0016898GCCUAGGAGUCCUUGGUCAGUGGGGACAUGGAGAAGGCUUCUGAGGA> hsa-mir-4532 MI0016899ACAGACCCCGGGGAGCCCGGCGGUGAAGCUCCUGGUAUCCUGGGUGUCUGA> hsa-mir-4533 MI0016900UGAGAAUGUGGAAGGAGGUUGCCGGACGCUGCUGGCUGCCUUCCAGCGUCCACUUCCCUUUCUCUCUCUCC> hsa-mir-4534 MI0016901UGUGAAUGACCCCCUUCCAGAGCCAAAAUCACCAGGGAUGGAGGAGGGGUCUUGGGUACU> hsa-mir-4535 MI0016903AACUGGGUCCCAGUCUUCACAGUUGGUUUCUGACACGUGGACCUGGCUGGGACGAUGUG > hsa-mir-4536-1 MI0016906AUGUGGUAGAUAUAUGCACGAUAUAUAUACUGCCCUGCUUUUAUACAUACAUACAUACAUACCUAUAUCGUGCAUAUAUCUACCACAU> hsa-mir-4536-2 MI0019149AUGUGGUAGAUAUAUGCACGAUAUAGGUAUGUAUGUAUGUAUGUAUAAAAGCAGGGCAGUAUAUAUAUCGUGCAUAUAUCUACCACAU> hsa-mir-4537 MI0016908UGAGCCGAGCUGAGCUUAGCUGGGCUGAGCUAACCAGGGCUGGGCUGAGCUGGGCUGAGCUGAGCUGAGC> hsa-m ir-4538 MI0016909GAGCUUGGAUGAGCUGGGCUGAACUGGGCUGGGUUGAGCUGGGCUGGGCUGAGUUGAGCCAGGCUGAUCUGGGCUGAG> hsa-mir-4539 MI0016910UGAGCUGGGCUCUGCUGUGCUGUGCUGAGCAGGGCUGAGCUGAACUGGGCUGAGCUGGGC> hsa-mir-4540 MI0016911AAGCUGCAUGGACCAGGACUUGGCACCUUUGGCCUUAGUCCUGCCUGUAGGUUUA> hsa-mir-4632 MI0017259GAGGGCAGCGUGGGUGUGGCGGAGGCAGGCGUGACCGUUUGCCGCCCUCUCGCUGCUCUAG> hsa-mir-4633 MI0017260UGGCAAGUCUCCGCAUAUGCCUGGCUAGCUCCUCCACAAAUGCGUGUGGAGGAGCUAGCCAGGCAUAUGCAGAGCGUCA> hsa-mir-4634 MI0017261GGACAAGGGCGGCGCGACCGGCCCGGGGCUCUUGGGCGGCCGCGUUUCCCCUCC> hsa-mir-4635 MI0017262CCGGGACUUUGUGGGUUCUGACCCCACUUGGAUCACGCCGACAACACUGGUCUUGAAGUCAGAACCCGCAAAGUCCUGG> hsa-mir -4636 MI0017263UAGAUUCAGAACUCGUGUUCAAAGCCUUUAGCCCAGCAAUGGGAGAGUGCUAAAGGCUUCAAGCACGAGUUCUGAAUCUA> hsa-mir-4637 MI0017264CCCUUACUUGGAUCUGCAAUUAGUAUUUUAAUCAUAGAUUGUAUUUAGUUAGUUUUUAAUACUAACUGCAGAUUCAAGUGAGGG> hsa-mir-4638 MI0017265GACUCGGCUGCGGUGGACAAGUCCGGCUCCAGAACCUGGACACCGCUCAGCCGGCCGCGGCAGGGGUC> hsa-mir-4639 MI0017266UUGCUAAGUAGGCUGAGAUUGAUGUCAGGUUAUCCCCAAGCAUAACCUCACUCUC ACCUUGCUUUGCAG> hsa-mir-4640 MI0017267CUGUGGGCUGGGCCAGGGAGCAGCUGGUGGGUGGGAAGUAAGAUCUGACCUGGACUCCAUCCCACCCACCCCCUGUUUCCUGGCCCACAG> hsa-mir-4641 MI0017268GGGGGGCAGGGGGCAGAGGGCAUCAGAGGACAGCCGCCUGGUGCCCAUGCCAUACUUUUGCCUCAG> hsa-mir-4642 MI0017269CACAACUGCAUGGCAUCGUCCCCUGGUGGCUGUGGCCUAGGGCAAGCCACAAAGCCACUCAGUGAUGAUGCCAGCAGUUGUG> hsa-mir-4643 MI0017270GUGUGCCCUAGCAUUUAUAAUCAUGUGUUCAUUCACAUGAUCAUAAGUGGACACAUGACCAUAAAUGCUAAAGCACAC> hsa-mir-4644 MI0017271GCGGCGGUGCUCUGCCUCUUUCUCCAUCCACCCUGGUCCAGGUCCACAGCAGUGGAGAGAGAAAAGAGACAGAAGGAUGGCCGU> hsa-mir-4645 MI0017272UGAUAGGGAAACCAGGCAAGAAAUAUUGUCUCCUCAAGUUGCGACGAGACAGUAGUUCUUGCCUGGUUUCUCUAUCA> hsa-mir-4646 MI0017273ACUGGGAAGAGGAGCUGAGGGACAUUGCGGAGAGGGUCUCACAUUGUCCCUCUCCCUUCCCAG > hsa-mir-4647 MI0017274CCAGGAGGGUGAAGAUGGUGCUGUGCUGAGGAAAGGGGAUGCAGAGCCCUGCCCAGCACCACCACCUCCUAUGCUCCUGG> hsa-mir-4648 MI0017275UGUGGGACUGCAAAUGGGAGCUCAGCACCUGCCUGCCACCCACGCAGACCAG > Hsa-mir-4650-1 MI0017277UUCUGUAGAGAUUAUCAGGCCUCUUUCUACCUUCCAAGGCUCAGAAGGUAGAAUGAGGCCUGACAUAUCUGCAGGA> hsa-mir-4650-2 MI0017278UUCUGUAGAGAUUAUCAGGCCUCUUUCUACCUUCCAAGGCUCAGAAGGUAGAAUGAGGCCUGACAUAUCUGCAGGA> hsa-mir-4651 MI0017279CGGCGACGGCGGGGUGGGUGAGGUCGGGCCCCAAGACUCGGGGUUUGCCGGGCGCCUCAGUUCACCGCGGCCG> hsa-mir-4652 MI0017280UAUUGGACGAGGGGACUGGUUAAUAGAACUAACUAACCAGAACUAUUUUGUUCUGUUAACCCAUCCCCUCAUCUAAUA> hsa-mir-4653 MI0017281UUGUCCAAUUCUCUGAGCAAGGCUUAACACCAAAGGGUUAAGGGUUUGCUCUGGAGUUAAGGGUUGCUUGGAGAAUUGGAGAA> hsa-mir-4654 MI0017282CUGGCUGGUUGUGGGAUCUGGAGGCAUCUGGGGUUGGAAUGUGACCCCAGUCUCCUUUUCCCUCAUCAUCUGCCAG> hsa-mir -4655 MI0017283CCAAGGGCACACCGGGGAUGGCAGAGGGUCGUGGGAAAGUGUUGACCCUCGUCAGGUCCCCGGGGAGCCCCUGG> hsa-mir-4656 MI0017284AGGCUGGCGUGGGCUGAGGGCAGGAGGCCUGUGGCCGGUCCCAGGCCUCCUGCUUCCUGGGCUCAGGCUCGGUUU> hsa-mir-4657 MI0017285AAUGUGGAAGUGGUCUGAGGCAUAUAGAGUAUAUGCCAAGAACACUACCAUAU> hsa-mir-4658 MI0017286GCUGCCCUUCACUCAGAGCAUCUACACCCACUACCGGUGAGUGUGGAUCCUGGAGGAAUCGUGGC> hsa-mir-4659a MI0017287GAAACUGCUGAAG CUGCCAUGUCUAAGAAGAAAACUUUGGAGAAAAAUUUUCUUCUUAGACAUGGCAACGUCAACAGUUUC> hsa-mir-4659b MI0017291CUGUUGACGUUGCCAUGUCUAAGAAGAAAAUUUUUCUCCAAAGUUUUCUUCUUAGACAUGGCAGCUUCAGCAG> hsa-mir-4660 MI0017288ACUCCUUCUGCAGCUCUGGUGGAAAAUGGAGAAGACUUUUCCUUUCCUCCAUCUCCCCCAGGGCCUGGUGGAGU> hsa-mir-4661 MI0017289UUUACUCUGAACUAGCUCUGUGGAUCCUGACAGACAGCCUGAUAGACAGGAUCCACAGAGCUAGUCCAGAGUAAA> hsa-mir-4662a MI0017290UCUAUUUAGCCAAUUGUCCAUCUUUAGCUAUUCUGAAUGCCUAAAGAUAGACAAUUGGCUAAAUAGA> hsa-mir-4662b MI0017293CACAAUUUCUAUUUAGCCAAUUGUCUAUCUUUAGGCAUUCAGAAUAGCUAAAGAUGGACAAUUGGCUAAAUAGACACUGUG> hsa-mir-4663 MI0017292CUGUGGUGGAGCUGAGCUCCAUGGACGUGCAGUGGCAUCUGUCAUUGCUGCCUUCCUGGAGCUCAGGCCCUUGCAG> hsa-mir-4664 MI0017294GUUGGGGGCUGGGGUGCCCACUCCGCAAGUUAUCACUGAGCGACUUCCGGUCUGUGAGCCCCGUCCUCCGC > hsa-mir-4665 MI0017295CUCGAGGUGCUGGGGGACGCGUGAGCGCGAGCCGCUUCCUCACGGCUCGGCCGCGGCGCGUAGCCCCCGCCACAUCGGG> hsa-mir-4666a MI0017296AUCACUUAAAUACAUGUCAGAUUGUAUGCCUACAAAAUCCCUCCAGGCAUACAAUC
UGACAUGUAUUUAAGAGAU> hsa-mir-4666b MI0019299UGUCUAAAUUGCAUGUCAGAUUGUAAUUCCCAGGCCCUUCCUCCAAUACUGGGAAUUACAAUUUGACAUGCAAUUUAGACA> hsa-mir-4667 MI0017297UGACUGGGGAGCAGAAGGAGAACCCAAGAAAAGCUGACUUGGAGGUCCCUCCUUCUGUCCCCACAG> hsa-mir-4668 MI0017298AGGGAAAAAAAAAAGGAUUUGUCUUGUAGCCAGGAUAUUGUUUUAAAGAAAAUCCUUUUUGUUUUUCCAG> hsa-mir-4669 MI0017300GCCUCCCUUCACUUCCUGGCCAUCCAGGCAUCUGUGUCUGUGUCCGGGAAGUGGAGGAGGGC> hsa-mir-4670 MI0017301CUCUAGGAAGCGACCAUGAUGUAACUUCACAGACUCUCCAAAAGUCUGAAGUUACAUCAUGGUCGCUUCCUAGAG> hsa-mir-4671 MI0017302UAUUUUAAGACCGAAGACUGUGCGCUAAUCUCUUAGCACUGAAGAUUAGUGCAUAGUCUUUGGUCUCAAAAUA> hsa-mir-4672 MI0017303GGCUGCUUCUCGCCUCUGUCCAGCUGUGUGGCCUUGGACAAGCCUCUUGGUUACACAGCUGGACAGAGGCACGAAACAGCC > hsa-mir-4673 MI0017304GUCCAGGCAGGAGCCGGACUGGACCUCAGGGAAGAGGCUGACCCGGCCCCUCUUGCGGC> hsa-mir-4674 MI0017305CCCAGGCGCCCGCUCCCGACCCACGCCGCGCCGCCGGGUCCCUCCUCCCCGGAGAGGCUGGGCUCGGGACGCGCGGCUCAGCUCGGG> hsa-mir-4675 MI0017306CAUGAGAAAUCCUGCUGGUCAACCAUAGCCCUGGUCAGACUCUCCGGGGCUGUGAUUGACCAGCAGGACUUCUCAUG> hsa-mir-4676 MI001 7307UGAAUGAAAGAGCCAGUGGUGAGACAGUGAGUUGAUUACUUCUCACUGUUUCACCACUGGCUCUUUGGUUCA> hsa-mir-4677 MI0017308GCAAAGCAGCAAUUGUUCUUUGGUCUUUCAGCCAUGACCUGACCUUCUGUCUGUGAGACCAAAGAACUACUUUGCUUGGC> hsa-mir-4678 MI0017309GGAAAAAACAAGGUAUUGUUCAGACUUAUGAUUUUUGGGGUCAAAGAUUCUGAGCAAUAACCUAUUAAAAAACC> hsa-mir-4679-1 MI0017310GUCUUUUUUCUGUGAUAGAGAUUCUUUGCUUUGUUAGAAACAAAAAGCAAAGAAUCUCUAUCACAGAAAAAAGAU> hsa-mir-4679-2 MI0017311UAUCUUUUUUCUGUGAUAGAGAUUCUUUGCUUUUUGUUUCUAACAAAGCAAAGAAUCUCUAUCACAGAAAAAAGACG> hsa-mir-4680 MI0017312UAUAAGAACUCUUGCAGUCUUAGAUGUUAUAAAAAUAUAUAUCUGAAUUGUAAGAGUUGUUAGCAC> hsa-mir-4681 MI0017313GGCAACGGGAAUGCAGGCUGUAUCUGCAGGGCAUUGUGCUAACAGGUGCAGGCUGCAGACCUGUCACAGGCC> hsa- mir-4682 MI0017314UGCCCCUGGUCUGAGUUCCUGGAGCCUGGUCUGUCACUGGGGAAGUCCAGAGCUCCAAGGCUCAGUGCCCAGGGGACGCA> hsa-mir-4683 MI0017315GACACGCAAGACGAGGCGGGCCUGGAGGUGCACCAGUUCUGGCCGCUGGUGGAGAUCCAGUGCUCGCCCGAUCUCAAGUUC> hsa-mir-4684 MI0017316GCACCAGGGGUACCUCUCUACUGACUUGCAACAUACAUUUGUCUUGGUGUGUUGCAAGUCGGUGGAGACGUACCCUUGGUGC> hsa-mir-4685 MI0017317UAG CCCAGGGCUUGGAGUGGGGCAAGGUUGUUGGUGAUAUGGCUUCCUCUCCCUUCCUGCCCUGGCUAG> hsa-mir-4686 MI0017318GGCUUCCUGUAUCUGCUGGGCUUUCUGGUGUUGGCAGCCCAAGAUGACACCCUGGGCCCAGCAGGAGCCAGAAGCC> hsa-mir-4687 MI0017319ACCUGAGGAGCCAGCCCUCCUCCCGCACCCAAACUUGGAGCACUUGACCUUUGGCUGUUGGAGGGGGCAGGCUCGCGGGU> hsa-mir-4688 MI0017321GUCUACUCCCAGGGUGCCAAGCUGUUUCGUGUUCCCUCCCUAGGGGAUCCCAGGUAGGGGCAGCAGAGGACCUGGGCCUGGAC> hsa-mir-4689 MI0017322GGUUUCUCCUUGAGGAGACAUGGUGGGGGCCGGUCAGGCAGCCCAUGCCAUGUGUCCUCAUGGAGAGGCC> hsa-mir-4690 MI0017323GAGCAGGCGAGGCUGGGCUGAACCCGUGGGUGAGGAGUGCAGCCCAGCUGAGGCCUCUGC> hsa-mir-4691 MI0017324GGAGCACUCCCAGGUCCUCCAGGCCAUGAGCUGCGGCCCUGAUGUCUCUACUCCAGCCACGGACUGAGAGUGCAUAGGAGUGUCC> hsa-mir-4692 MI0017325GUUUUACUUGAUACCCACACUGCCUGGGUGGGACACUCAGGCAGUGUGGGUAUCAGAUAAAAC > hsa-mir-4693 MI0017326GUUUAAAGAAUACUGUGAAUUCACUGUCAAAUUCAAAUAAAGUGAGAGUGGAAUUCAGUAUUUAAGGAAU> hsa-mir-4694 MI0017327CAAAUACAUAGGUGUUAUCCUAUCCAUUGCCUCUCUCUCAGA AGCCCAAUGCCAUGGGCCUGAUCUCACCGCUGCCUCCUUCCC> hsa-mir-4696 MI0017329CAAAGCCACUGCAAGACGGAUACUGUCAUCUAUUCCAGAAGAUGACAAUGUCCAUUUUGCAGUGGCUUUG> hsa-mir-4697 MI0017330GGGCCCAGAAGGGGGCGCAGUCACUGACGUGAAGGGACCACAUCCCGCUUCAUGUCAGUGACUCCUGCCCCUUGGUCU> hsa-mir-4698 MI0017331UGCUUCUCCUGGGGUCUUCCUCUACAUUUCCACCUAGACGGGCCUGGGUCAAAAUGUAGAGGAAGACCCCAGAAGGAGCA> hsa-mir-4699 MI0017332AGCAAUUGGAGAAGAUUGCAGAGUAAGUUCCUGAUUAAGAAAUGGAAUUUACUCUGCAAUCUUCUCCAAUUGCU> hsa-mir-4700 MI0017333UCAGUGAGGUCUGGGGAUGAGGACAGUGUGUCCUGAAAUUCACAGGACUGACUCCUCACCCCAGUGCACGAGGA> hsa-mir-4701 MI0017334CCUUGGCCACCACACCUACCCCUUGUGAAUGUCGGGCAAUGGGUGAUGGGUGUGGUGUCCACA> hsa-mir-4703 MI0017336UUAUGCAUAUUAGCAAUACAGUACAAAUAUAGUGUGUUUGAUUUGCACUGUAGUUGUAUUGUAUUGCCACUCUGUAUAA > hsa-mir-4704 MI0017337CUUAUCCUAGACACUAGGCAUGUGAGUGAUUGUCUUCCUCACUCAAUCAGUCACAUAUCUAGUGUCUAGAAUGAG> hsa-mir-4705 MI0017338CUCACAAGAUCAAUCACUUGGUAAUUGCUGUGAUAACAACUCAGCAAUUACCAAG UGUCCUGGGCUCUGUGGU> hsa-mir-4707 MI0017340GGUUCCGGAGCCCCGGCGCGGGCGGGUUCUGGGGUGUAGACGCUGCUGGCCAGCCCGCCCCAGCCGAGGUUCUCGGCACC> hsa-mir-4708 MI0017341UUUAGGAGAGAGAUGCCGCCUUGCUCCUUGAACAGGAGGAGCAAGGCGGCAUCUCUCUGAUACUAAA> hsa-mir-4709 MI0017342CUGCUUCAACAACAGUGACUUGCUCUCCAAUGGUAUCCAGUGAUUCGUUGAAGAGGAGGUGCUCUGUAGCAG> hsa-mir-4710 MI0017344GACCGAGUGGGGUGAGGGCAGGUGGUUCUUCCCGAAGCAGCUCUCGCCUCUUCGUC> hsa-mir-4711 MI0017345AAAUGUGCAUCAGGCCAGAAGACAUGAGCCCUUUGGAAAGGUCUCGUGUCUUCUGGCUUGAUGCACAUUU> hsa-mir-4712 MI0017346GACAGGAUUCCAGUACAGGUCUCUCAUUUCCUUCAUGAUUAGGAAUACUACUUUGAAAUGAGAGACCUGUACUGUAUCUGUU> hsa-mir-4713 MI0017347GUCCCCAUUUUUCUCCCACUACCAGGCUCCCAUAAGGGUCGAAUGGGAUCCAGACAGUGGGAGAAAAAUGGGGAC > hsa-mir-4714 MI0017348AUUUUGGCCAACUCUGACCCCUUAGGUUGAUGUCAGAAUGAGGUGUACCAACCUAGGUGGUCAGAGUUGGCCAAAAU> hsa-mir-4715 MI0017349GGGGAAUGAAAGUUGGCUGCAGUUAAGGUGGCUAAUCAGCUGAUGGUGCCACCUUAACUGCAGCCAAUUCUAAUUCCCC> hsa-mir-4716 MI0017350CAUACUUUGUCUCCAUGUUUCCUUCCCCCUUCUGUAUACAUGUAUACAGGAGGAAGGGGGAAGGAAACAUGGAGACAAAGUGUG> hsa-mir-4 717 MI0017352GGCAGUGUUUAGGCCACAGCCACCCAUGUGUAGGGGUGGCUACACAUGGGUGGCUGUGGCCUAAACACUGCC> hsa-mir-4718 MI0017353AGCUGUACCUGAAACCAAGCACCUGUUUGUGACUUGGCUUCAGUUACUAGC> hsa-mir-4719 MI0017354ACAAUGAUGACUUGUAUGUUAUAGAUUUGUGAUUACAUUAAAACUUAAAAUUUCACAAAUCUAUAAUAUGCAGGUCAUCACUGU> hsa-mir-4720 MI0017355AAGCCUGGCAUAUUUGGUAUAACUUAAGCACCAGGUAAAAUCUGGUGCUUAAGUUGUACCAAGUAUAGCCAAGUUU> hsa-mir-4721 MI0017356GGGCCUGGUCAUGGUCAAGCCAGGUUCCAUCAAGCCCCACCAGAAGGUGGAGGCCCAGGUGAGGGCUCCAGGUGACGGUGGGCAGGGUU> hsa-mir-4722 MI0017357GGCAGGAGGGCUGUGCCAGGUUGGCUGGGCCAGGCCUGACCUGCCAGCACCUCCCUGCAG> hsa-mir-4723 MI0017359AGUUGGUGGGGGAGCCAUGAGAUAAGAGCACCUCCUAGAGAAUGUUGAACUAAAGGUGCCCUCUCUGGCUCCUCCCCAAAG> hsa-mir-4724 MI0017361ACGCAAAAUGAACUGAACCAGGAGUGAGCUUCGUGUACAUUAUCUAUUAGAAAAUGAAGUACCUUCUGGUUCAGCUAGUCCCUGUGCGU> hsa-mir-4725 MI0017362GUGUCUCUCUGGAGACCCUGCAGCCUUCCCACCCACCAGGGAGCUUUCCAUGGGCUGUGGGGAAGGCGUCAGUGUCGGGUGAGGGAACAC> hsa-mir-4726 MI0017363AGGGCCAGAGGAGCCUGGAGUGGUCGGGUCGACUGAACCCAGGUUCCCUCUGGCCGCA> hsa-mir-4727 MI0017364AAUCUGC CAGCUUCCACAGUGGCAGAUUUUCCCAUAGUGGGAAGCUGGCAGAUUC> hsa-mir-4728 MI0017365GUGGGAGGGGAGAGGCAGCAAGCACACAGGGCCUGGGACUAGCAUGCUGACCUCCCUCCUGCCCCAG> hsa-mir-4729 MI0017366UCUGUUUCCUCAUUUAUCUGUUGGGAAGCUAACUGUGACCUUAGCGUCCCAGCAGAUAAAUGAGGAAACAGA> hsa-mir-4730 MI0017367CGCAGGCCUCUGGCGGAGCCCAUUCCAUGCCAGAUGCUGAGCGAUGGCUGGUGUGUGCUGCUCCACAGGCCUGGUG> hsa-mir-4731 MI0017368CCCUGCCAGUGCUGGGGGCCACAUGAGUGUGCAGUCAUCCACACACAAGUGGCCCCCAACACUGGCAGGG> hsa-mir-4732 MI0017369GAGGGAGCUGUAGAGCAGGGAGCAGGAAGCUGUGUGUGUCCAGCCCUGACCUGUCCUGUUCUGCCCCCAGCCCCUC> hsa-mir-4733 MI0017370GGUCGCUUAAAUCCCAAUGCUAGACCCGGUGGCAAUCAAGGUCUAGCCACCAGGUCUAGCAUUGGGAUUUAAGCCC> hsa-mir-4734 MI0017371CUCGGGCCCGACCGCGCCGGCCCGCACCUCCCGGCCCGGAGCUGCGGGCUGCGGUCAGGGCGAUCCCGGG > hsa-mir-4735 MI0017372UGCAGUGCCUAAUUUGAACACCUUCGGUAUUCAUCAAAAUACCAAAGGUGCUCAAAUUAGACAUUGCA> hsa-mir-4736 MI0017373AGGCAGGUUAUCUGGGCUGCCAUCUCACUGGCUGCUUGCCUGCCU> hsa-mir-4737 17376GGUCGCAUUUCUCCUUCUUACCAGCGCGUUUUCAGUUUCAUAGGGAAGCCUUUCCAUGAAACUGGAGCGCCUGGAGGAGAAGGGGCC> hsa-mir-4739 MI0017377GGGAGGAAGAAGGGAGGAGGAGCGGAGGGGCCCUUGUCUUCCCAGAGCCUCUCCCUUCCUCCCCUCCCCCUCCC> hsa-mir-4740 MI0017378GCCAAGGACUGAUCCUCUCGGGCAGGGAGUCAGAGGGGACCGCCCGAGAGGAUCCGUCCCUGC> hsa-mir-4741 MI0017379CGGGCGGGGCGGGUCCGGCCGCCUCCGAGCCCGGCCGGCAGCCCCCGGCCUUAAAGCGCGGGCUGUCCGGAGGGGUCGGCUUUCCCACCG> hsa-mir-4742 MI0017380UCAGGCAAAGGGAUAUUUACAGAUACUUUUUAAAAUUUGUUUGAGUUGAGGCAGAUUAAAUAUCUGUAUUCUCCUUUGCCUGCAG> hsa-mir-4743 MI0017381GCUGGCCGGAUGGGACAGGAGGCAUGAAUGAGCCAUCUUUCCAAUGCCUUUCUGUCUUUUCUGGUCCAG> hsa-mir-4744 MI0017382GUAAUCACAUCUAAAGACUAGACUUCGCUAUGACCAGGCCAUAGUAAACAUCAUAGUAUGUCUAGUCUUUAGGUUUGAUUAC> hsa-mir-4745 MI0017384GUGAGUGGGGCUCCCGGGACGGCGCCCGCCCUGGCCCUGGCCCGGCGACGUCUCACGGUCCC > hsa-mir-4746 MI0017385GUGUCUGUGCCGGUCCCAGGAGAACCUGCAGAGGCAUCGGGUCAGCGGUGCUCCUGCGGGCCGACACUCAC> hsa-mir-4747 MI0017386AGGGAAGGAGGCUUGGUCUUAGCACGGGGUCUAAGCCCGGGCUUUCCUCCCAG CUGGUGCUAGAGAGGAAAGCAGACCCUACCCAACCCCACGCCCUACUACAGCCA> hsa-mir-4749 MI0017388CCUGCGGGGACAGGCCAGGGCAUCUAGGCUGUGCACAGUGACGCCCCUCCUGCCCCCACAG> hsa-mir-4750 MI0017389CGCUCGGGCGGAGGUGGUUGAGUGCCGACUGGCGCCUGACCCACCCCCUCCCGCAG> hsa-mir-4751 MI0017390CCCGGAGCCAGAGGACCCGUAGCUGCUAGAAGGGCAGGGGUGUGGCUUCUGGGGGCUGGUCUUCAGCUCUGGCG> hsa-mir-4752 MI0017391AGUGUCUCCUUGUGGAUCUCAAGGAUGUGCUUCCACAUAGCAGCAUGUUCUUCAGAUGGACAAGGAGACACU> hsa-mir-4753 MI0017392AUAUCUACACAAGGCCAAAGGAAGAGAACAGAUAUAUCCACAGUACACUUGGCUGUUCUCUUUCUUUAGCCUUGUGUAGAUAU> hsa-mir-4754 MI0017394ACGCGCCUGAUGCGGACCUGGGUUAGCGGAGUGAGGCCCAGUGGUCACCGCCGCCCUCCGCAGGUCCAGGUUGCCGUGCGCAUGUGCCU> hsa-mir-4755 MI0017395AGAUUCAGCUUUCCCUUCAGAGCCUGGCUUUGGCAUCUAUGAAAGCCAGGCUCUGAAGGGAAAGUUGAAUCU > hsa-mir-4756 MI0017397GGGAUAAAAUGCAGGGAGGCGCUCUCUCUGCUGCCGAUUCUGCACCAGAGAUGGUUGCCUUCCUAUAUUUGUGUC> hsa-mir-4757 MI0017398UUCCAGCCCGAGGCCUCUGUGACGUCACGGUGUCUGCGGGAGGAG UGCUGACCACCCUCCCC> hsa-mir-4759 MI0017400CAUUUAGGACUAGAUGUUGGAAUUAGACAGAAAAAAGUUAGACACAAAAAAUUGUGUCUAAUUCCAACAUCUAGUCCUAAAUG> hsa-mir-4760 MI0017401GCCAUGGUGUUUAGAUUGAACAUGAAGUUAGAAUUCUUAAGUAUCAAAACUAAAUUCAUGUUCAAUCUAAACCCCAUGGC> hsa-mir-4761 MI0017402GGACAAGGUGUGCAUGCCUGACCCGUUGUCAGACCUGGAAAAAGGGCCGGCUGUGGGCAGGGAGGGCAUGCGCACUUUGUCC> hsa-mir-4762 MI0017403CUGAUACCCCAAAUCUUGAUCAGAAGCCUUGAUCAGAAGCUAGGAAGGCUUCUGAUCAAGAUUUGUGGUGUCAAG> hsa-mir-4763 MI0017404CCUGUCCCUCCUGCCCUGCGCCUGCCCAGCCCUCCUGCUCUGGUGACUGAGGACCGCCAGGCAGGGGCUGGUGCUGGGCGGGGGGCGGCGGG> hsa-mir-4764 MI0017405UCUUCCCCAUGGAUGUGGAAGGAGUUAUCUGUCACCAGUCAGAUAACUGUCACCAGUCAGUUAACUCCUUUCACACCCAUGGGGAAGA> hsa-mir-4765 MI0017406UGGUGAUUUUGAACGUAGCUAUCCACCACUCAGCCUGGAAAAAGCUGAGUGAUUGAUAGCUAUGUUCAAAAUCACCA > hsa-mir-4766 MI0017407CUGAAGCUCCUUCUGAAAGAGCAGUUGGUGUUUAUUUUUACUAAAUAGCAAUUGCUCUUUGGAAGGAACUUGAG> hsa-mir-4767 MI0017408ACAUGGGCCCGCGGGCGCUCCUGGCCGCCGCCCGACUUCGGGGCCAG
GCGCGCGGGAGCCCGAGCGU> hsa-mir-4768 MI0017409AAACUUUGAUUCUCUCUGGAUCCCAUGGAUAUGGGAACUGUGAUGUCCAGGAGAUCCAGAGAGAAUCAGAGUUU> hsa-mir-4769 MI0017410GAGGAGAGGUGGGAUGGAGAGAAGGUAUGAGCUAAAAAUCCCCAAGCUCUGCCAUCCUCCCUCCCCUACUUCUCCCC> hsa-mir-4770 MI0017411GAGUUAUGGGGUCAUCUAUCCUUCCCUUGGAAAAUGAUCUGAGAUGACACUGUAGCUC> hsa-mir-4771-1 MI0017412GCUCUAGCCUAAUUUUAGAUCUGGUCUGCUUCAGUUUCACUCCAAGCAGACUUGACCUACAAUUAGCCUAGAGC> hsa-mir-4771-2 MI0017413GCUCUAGCCUAAUUUUAGAUCUGGUCUGCUUCAGUUUCACUCCAAGCAGACUUGACCUACAAUUAGCCUAGAGC> hsa-mir-4772 MI0017414GUGAUUGCCUCUGAUCAGGCAAAAUUGCAGACUGUCUUCCCAAAUAGCCUGCAACUUUGCCUGAUCAGAGGCAGUCAC> hsa- mir-4773-1 MI0017415UGCUCCCCAGCCUUUCUAUGCUCCUGUUCUGCUUUAUUUCAUCAAAGCAGAACAGGAGCAUAGAAAGGCUGGGGAGCA> hsa-mir-4773-2 MI0017416UGCUCCCCAGCCUUUCUAUGCUCCUGUUCUGCUUUGAUGAAAUAAAGCAGAACAGGAGCAUAGAAAGGCUGGGGAGCA> hsa-mir-4774 MI0017417UAUAUUGUUGUCUGGUAUGUAGUAGGUAAUAACUGACAAACAGACAAUUGCCUAACAUGUGCCAGAAAACAACAUA> hsa-mir-4775 MI0017418AUUAAGCUUUUAAUUUUUUGUUUCGGUCACUCUUGAUAGCAGACAUUGACUGAAACAAAAAAUUAAAAGCUUUAU > Hsa-mir-4776-1 MI0017419CUAUAUGCAGUGGACCAGGAUGGCAAGGGCUCUCCUGAAAGGACAGUAGAGCCCUUGCCAUCCUGGUCCACUGCAUAUAG> hsa-mir-4776-2 MI0017420CUAUAUGCAGUGGACCAGGAUGGCAAGGGCUCUACUGUCCUUUCAGGAGAGCCCUUGCCAUCCUGGUCCACUGCAUAUAG> hsa-mir-4777 MI0017421UAGAAUAUUUCGGCAUUCUAGAUGAGAGAUAUAUAUAUACCUCAUAUGUAUAUGGUAUACCUCAUCUAGAAUGCUGUAAUAUUCUA> hsa-mir-4778 MI0017422UCACAUGUCCAAUUCUGUAAAGGAAGAAGAGGUAAGAAGAAGUGAAGCCCUCUUCUUCCUUUGCAGAGUUGAAUAUGUGG> hsa-mir-4779 MI0017423UAAAUGUCUUACUGCUUUUACUGUUCCCUCCUAGAGUCCAUUCUUUACUCUAGGAGGGAAUAGUAAAAGCAGUAAGACAUUUA> hsa-mir-4780 MI0017424GGCCAGUGCCAGGGGGUCAGGCUCAAGGACCAGCCCAAAGGCCAGGCCUGACCCUUGAGCCUGAUCCCUAGCACUGAUCCC> hsa-mir -4781 MI0017426AGGUGCACGCUCUAGCGGGGAUUCCAAUAUUGGGCCAAUUCCCCCAAUGUUGGAAUCCUCGCUAGAGCGUGCACUU> hsa-mir-4782 MI0017427AUUGCCCAGUUCUGGAUAUGAAGACAAUCAAGAAAAGAUUUGGUGUUCUUGAUUGUCUUCAUAUCUAGAACUGGGCAGU> hsa-mir-4783 MI0017428GGGAAAGCGGAGGGCGCGCCCAGCUCCCGGGCUGAUUGCGCUAACAGUGGCCCCGGUGUUGGGGCGCGUCUGCCGCUGCCCC> hsa-mir-4784 MI0017429UGACUGGGCUGAGGAGAUGCUGGGACUGAGAGUGUCAUG GUGGAGCCUCCGUCCCUGCUCAUCCUCUCCGCAUGUUG> hsa-mir-4785 MI0017430GUAGGUGGGGACGCGGCGGCGCUGCUCCUCCGCUGCCGCCGGGAGAGUCGGCGACGCCGCCAGCUCCGCGCGC> hsa-mir-4786 MI0017433GGGCAUGGCCUGAGACCAGGACUGGAUGCACCACUCUCCCUGUGAUGAGGUGAAGCCAGCUCUGGUCUGGGCCAUUUCAC> hsa-mir-4787 MI0017434CGGUCCAGACGUGGCGGGGGUGGCGGCGGCAUCCCGGACGGCCUGUGAGGGAUGCGCCGCCCACUGCCCCGCGCCGCCUGACCG> hsa-mir-4788 MI0017435AAUGAAGGAUUACGGACCAGCUAAGGGAGGCAUUAGGAUCCUUAUUCUUGCCUCCCUUAGUUGGUCCCUAAUCCUUCGUU> hsa-mir-4789 MI0017436CAUGCUACGUAUGUAUACACCUGAUAUGUGUAUGUGUAAAUACAUAUCCACACACAUAGCAGGUGUAUAUAUAGGUAGCCUG> hsa-mir-4790 MI0017437CAAUGUGACAUCGCUUUACCAUUCAUGUUCACUGAAAGGUAGAUUUUAAAAACAUGAAUGGUAAAGCGAUGUCACAUUG> hsa-mir-4791 MI0017438UAAGAACUGGAUAUGAUGACUGAAAUAAGCUCCAUAUCAAUGAGAAUUUCAAUGGGAUUAUGUGCAGUCAAUGUCCAGUAAUUA > hsa-mir-4792 MI0017439GCAGCCCGGUGAGCGCUCUGGCCUGGCAGUGCGUCGGAAGAACAGGGCGGGUGGGGCCGCGCACAUCUCUGC> hsa-mir-4793 MI0017440UUUCUCCUCGCUGCCCGCACAUCCUGCUCCAGGCCAGAGGGAGGCCAAAGACCUCUG CUGGCUAUCUCACGAGACUGUAUGUCCUAACAGUGCUUGUAGUCUCAUGAGAUAGCCAGAUGUUAAAA> hsa-mir-4795 MI0017442UGAUAUGGAAGAAAUCCAGAAGUGGCUAAUAAUAUUGACACUAUAACAAUAAUGUCAAUAUUAUUAGCCACUUCUGGAUUUAUGAAUCA> hsa-mir-4796 MI0017443UAAAUUUGUGUCUAUACUCUGUCACUUUACUUUUGGCCUCAAGUCAUUGCAGUAAAGUGGCAGAGUAUAGACACAAAUUUA> hsa-mir-4797 MI0017444GACUCAGAAGACAGAGUGCCACUUACUGAAAGGUUUUUUCUCUCAGUAAGUGGCACUCUGUCUUCUGAGUU> hsa-mir-4798 MI0017445AAGUACAACUUCGGUAUACUUUGUGAAUUGGCUUUUACAAAAGACCAACUCACGAAGUAUACCGAAGUCAUACUU> hsa-mir-4799 MI0017446ACUGCUAAUAUCUAAAUGCAGCAUGCCAGUCCUGAGAUGCAGGGACUGGCAUGCUGCAUUUAUAUAUUAGCAGU> hsa-mir-4800 MI0017448GGAGAAAGGAGUGGACCGAGGAAGGAAGGAAGGCAAGGCUGUCUGUCCAUCCGUCCGUCUGUCCACCUACCUGUCAGUCC> hsa-mir-4801 MI0017449UUGAGGCUUGGUUUUCUUAUGUGUAAAAUGUAAUAACAUUUCUUAUGUUUAAAACACUUUACACAAGAAAACCAAGGCUCAA > hsa-mir-4802 MI0017450CUGACUGGCUUGUAUGGAGGUUCUAGACCAUGUUAGUGUUCAAGUCUACAUGGAUGGAAACCUUCAAGCAGGCCAAGCAG> hsa-mir-4803 MI0017451AGUGGGAUUUAACAUAAUAGUGUGGAUUGAAUCACACACACAUUCAACCCA 7452UCAGUGUAUUUGGACGGUAAGGUUAAGCAAGGUGCGUCGUAUCUUGCUUAACCUUGCCCUCGAAAUACACUGA> hsa-mir-4999 MI0017865AUAGAAAAUAAAACACAUACUGCUGUAUUGUCAGGUAGUGAUAGGAUUUAUCACUACCUGACAAUACAGUAUGUGUUUGUUUUAUAUAUUU> hsa-mir-5000 MI0017866CUGAAGAGUAGAGUGUGUGGUCCCAGUUCAGAAGUGUUCCUGAGUAACUUGUGCUUAUAACUCAGGACACUUCUGAACUUGGACCAUACAGGUCUCCCUGCUU> hsa-mir-5001 MI0017867AGCUCAGGGCGGCUGCGCAGAGGGCUGGACUCAGCGGCGGAGCUGGCUGCUGGCCUCAGUUCUGCCUCUGUCCAGGUCCUUGUGACCCGCCCGCUCUCCU> hsa-mir-5002 MI0017868UCUUCCUCUCUGUCCUCUGGAAUUUGGUUUCUGAGGCACUUAGUAGGUGAUAGCAUGACUGACUGCCUCACUGACCACUUCCAGAUGAGGGUUACUC> hsa-mir-5003 MI0017869AUGAGUUUGCUUUGUGUCAUCCUCACAACAACCUUGCAGGGUAGAGAUGAUUUUUCCUACUUUUCUAGGUUGUUGGGGGCUGGGGCAGGGGGAACAGAG> hsa-mir-5004 MI0017870GGCACUUGCUUGGGGGUUAGUGAGGACAGGGCAAAUUCACGAGAUUGGGUUGUGCAGAGGCUGACACUUGGAUUUUCCUGGGCCUCAGGACUUCCUUUCAGACAUGG> hsa-mir-5006 MI0017873AACCAUUAGGGGGCUGUGGUUUGCCAGGGCAGGAGGUGGAAGGGAGCCCCAUUUACAGUGGUAACUUCCUUUCCCUUUCCAUCCUGGCAGGCUUCAGAGAACUUUACCAG > hsa-mir-5007 MI0017874GGUAAACCUUGGUGACUAAUUAGAGUCUGGCU GAUAUGGUUUGACACAGAGCUAAAUCAUAUGAACCAAACUCUAAUUAGUCAAUAAUUUCUGUU> hsa-mir-5008 MI0017876GGGCUGACCCCUAGGGUCAGGUGAGGCCCUUGGGGCACAGUGGUGCCAUCUCCCCUGUGCUCCCAGGGCCUCGCCUGUCCCUUGAGGUCGGCCC> hsa-mir-5009 MI0017877GACCAGAAGUGUUUUGGAUUUUGGACUUUUUCAGAUUUGGGGAUAUUUGCAUUAUACUUAUCCUAAAUCUGAAAGUCCAAAACCUGAAAUGACCAAUAAG> hsa-mir-5010 MI0017878GAUCCAGGGAACCCUAGAGCAGGGGGAUGGCAGAGCAAAAUUCAUGGCCUACAGCUGCCUCUUGCCAAACUGCACUGGAUUUUGUGUCUCCCAUUCCCCAGAGCUGUCUGAGGUGCUUUG> hsa-mir-5011 MI0017879AGAUGGUAUUGAGUGGAUGCUGUUAUAUAUACAGCCAUGCACUCUGUAGUUUGGGUACACAGUGCAUGGCUGUAUAUAUAACACUAUCCAUUCAUCUUUCAGC> hsa-mir-5047 MI0017932GAAGCGCUUGCCUAGACGAGACACAGUGCAUAAAAACAACUUUUGGGGGACAGGUAUGUUUUCUUGCAGCUGCGGUUGUAAGGUCUUGGCAAGACAAGCA> hsa-mir-5087 MI0017976AGCUUUCUACGGGUUUGUAGCUUUGCUGGCAUGUUAAGUGUUGUCCUACAGUCGCAAGCAUAAGAAAGAGAAAGUA> hsa-mir-5088 MI0017977UCUCCAGGGUGGCCAGGCGGGGCCGGGCCUGAGGGAUGGAGGGGAGCCCAUCAGGGCUCAGGGAUUGGAUGGAGGUGAUGGGGG > hsa-mir-5089 MI0017978AAGGACUUCAGUGGGAUUUCUGAGUAGCAUCCUUGGAAUCUGCACUCAAGGGAUGCUACGGAAAUCCCACUGAA GUCCUUUU> hsa-mir-5090 MI0017979UCUGAGGUACCCGGGGCAGAUUGGUGUAGGGUGCAAAGCCUGCCCGCCCCCUAAGCCUUCUGCCCCCAACUCCAGCCUGUCAGGA> hsa-mir-5091 MI0017980GACUGUGGCGACGGAGACGACAAGACUGUGCUGGUCGCGGGUUGUGGGGUUUAGGUCACCGGCAGGGGUCUGGAGUCCCUGGAGGUUAGGGCU> hsa-mir-5092 MI0017981AUCCCAGAUCAGAUGCCAAAGCCAGUGGGGACUGGACAACAUGAUGAGCCCAAACCAAUCCACGCUGAGCUUGGCAUCUGAUUUGGGA> hsa-mir-5093 MI0017982CCCGCCAGGUCCACAUGCCAGAGUGUCAACGUGACCCAGCCAGCCUCCUUCCUGAGCUAGGAGGAUUAGGAAAUGAGGCUGGCUAGGAGCACAGCCAGGG> hsa-mir-5094 MI0017983AAAAGAAAAAAAUCAGUGAAUGCCUUGAACCUAACACACUGCCUUUUAUGUGGUAGGUACAGUGGGCUCACUGAAACAUUCAACU> hsa-mir-5095 MI0018001CUGGGAUUACAGGCGUGAACCACCGCGCCCGGCCUAACUUUUAAGAAACGUCGGCCCGGGAGCGGUGGCUCACGCCUGUAAUCCCAGC> hsa-mir-5096 MI0018004AGUAGAGGUGGGGUUUCACCAUGUUGGUCAGGCUGGUCUCAAACUCCUGACCUCAGGUGAUCCAUCCACC > hsa-mir-5100 MI0019116CCAUGAGGAGCUGGCAGUGGGAUGGCCUGGGGGUAGGAGCGUGGCUUCUGGAGCUAGCCACAUGGGUUCAGAUCCCAGCGGUGCCUCUAACUGGCCACAGGACCUUGGGCAGUCAGCU> hsa-mir-5186 MI0018165UCAGCCAGCUUAUGA AUUUCUUUCUGGGGAGAGAUUGGUAGAAAUCAGGUGAGAGGGUCAUGCCAUAAGCUGGCUAAC> hsa-mir-5187 MI0018166GACUAAGGGUGGGAUGAGGGAUUGAAGUGGAGCAGGAAUGCGCUUUUCUCCACUGAAUCCUCUUUUCCUCAGGUGG> hsa-mir-5188 MI0018167GGGAGGCAUGGAAAUUUCUCUGGUUUCAAUGGGUACGAUUAUUGUAAGCAGGAUCCAUUCAAUAAUCGGACCCAUUUAAACCGGAGAUUUUAAAAGACAGGAAUAGAAUCCCA> hsa-mir-5189 MI0018168GGCCCGCCUUUUAGGGGCCUCGCUGUCUGGGCACAGGCGGAUGGACAGGCUGGCCUCUGGAUGACCUGCCAACCGUCAGAGCCCAGACCCACGUGGCCUCAGUUGGGGACCAGG> hsa-mir-5190 MI0018169GGUCAUACCCUGGCUCCAGCCCUGUCACAUGGUUAAUGUUCCACAGCCAGUGACUGAGCUGGAGCCAGGGCCACUGCCCC> hsa-mir-5191 MI0018170AGUUGGCCAGGACCCCAAGCCCCCAGCACUUCAUUCUUGCUGUCCUCUCCUGGUCUGGGAGGAUAGAAGAGAGGAUAGGAAGAAUGAAGUGCUGGGCGCUUAGGGGGAUCCUGGCCAACU> hsa-mir-5192 MI0018171UUAGUUCCAGCCUCCUGGCUCACCUGGAACCAUUUCUCCUGGGAAGCAUGGUAGCCAGGAGAGUGGAUUCCAGGUGGUGAGGGCUUGGUACU> hsa-mir-5193 MI0018172CCUAGGAAAGGCUGCUGGUAACUGGGAUGGGGGUUGGGGGGAGGUAAGAAGUCUCUGACUCCUCCUCUACCUCAUCCCAGUUCCAUCACCUGAAGUGGACCUCUUGGGA > hsa-mir-5194 MI0018173AUUUCUUUGGGUUAACUUAAACUCAGCCCUUCUAGGCCCAUUCUUUCA CUCAGGAAUUGGAUAAGCUUUUCUGAGGGGUUUGGAAUGGGAUGGCAGGGAGAGUCACCAGACACCAUGAA> hsa-mir-5195 MI0018174GAGCAAAAACCAGAGAACAACAUGGGAGCGUUCCUAACCCCUAAGGCAACUGGAUGGGAGACCUGACCCAUCCAGUUCUCUGAGGGGGCUCUUGUGUGUUCUACAAGGUUGUUCA> hsa-mir-5196 MI0018175UCUGAGGAGACCUGGGCUGUCAGAGGCCAGGGAAGGGGACGAGGGUUGGGGAACAGGUGGUUAGCACUUCAUCCUCGUCUCCCUCCCAGGUUAGAAGGGCCCCCCUCUCUGAAGG> hsa-mir-5197 MI0018176UAUGGGAUUCCACAGACAAUGAGUAUCAAUGGCACAAACUCAUUCUUGAAUUUUUGCCAGUUCAAGAAGAGACUGAGUCAUCGAAUGCUCUAAAUGUCACUUCACCUCAUGU> hsa-mir-5571 MI0019115AUCUGACACAAAAUGUGAACCAAGCAAUUCUCAAAGGAGCCUCCCAGGAAAUUCACUUUAGGAAGUCCUAGGAGGCUCCUCUGAGAGUUGCUAAAACAAAACAUUGAGAGUCC> hsa-mir-5572 MI0019117AGCCAGACAAGAGGGUCAUGGGGAGUCACUGUCAACCCAGAGCAGGCACUGCCCCUGCGACCAGCCUGGGGCAUCGGUUGGGGUGCAGGGGUCUGCUGGUGAUGCUUUCCAUCUCUUUGCUUUGUCCUGAUUGUAGC> hsa-mir-5579 MI0019133UAUGGUACUCCUUAAGCUAACAGGCCCCUGUCACCAUUAGCUUAAGGAGUACCAGAUC> hsa-mir-5580 MI0019135UGCUGGCUCAUUUCAUAUGUGUGCUGAGAAAAUUCACACAUAUGAAGUGAGCCAGCAC > hsa-mir-5581 MI0019136AGCCUUCCAGGAGAAAUGGAGACCCUAUACAUACCUG UUUCCAUGCCUCCUAGAAGUUCC> hsa-mir-5582 MI0019138UAGGCACACUUAAAGUUAUAGCUACAUCAGUUAUAACUAUAUCAGUUAAAACUUUAAGUGUGCCUAGG> hsa-mir-5583-1 MI0019139AAACUAAUAUACCCAUAUUCUGGCUAGGUGAUCAUCAGAAUAUGGGUAUAUUAGUUUGG> hsa-mir-5583-2 MI0019140AAACUAAUAUACCCAUAUUCUGAUGAUCACCUAGCCAGAAUAUGGGUAUAUUAGUUUGG> hsa-mir-5584 MI0019141CAGGGAAAUGGGAAGAACUAGAUUUGAAUCCAGACCUUUAGUUCUUCCCUUUGCCCAAUU> hsa-mir-5585 MI0019142UGAAGUACCAGCUACUCGAGAGGUCAGAGGAUUGCUCCUGAAUAGCUGGGACUACAGGU> hsa-mir-5586 MI0019143UAUCCAGCUUGUUACUAUAUGCUUUUUAAAUGGGGCACAGAGUGACAAGCUGGUUAAAG> hsa- mir-5587 MI0019144AUGGUCACCUCCGGGACUCAGCCCUGUGCUGAGCCCCGGGCAGUGUGAUCAUC> hsa-mir-5588 MI0019147ACUGGCAUUAGUGGGACUUUUUUUUUUUUUUUUUUUAAUGUUAAAAGUCCCACUAAUGCCAGC> hsa-mir-5589 MI0019148GGCUGGGUGCUCUUGUGCAGUGAGCAACCUACACAACUGCACAUGGCAACCUAGCUCCCA> hsa-mir-5590 MI0019150UUGCCAUACAUAGACUUUAUUGUGUUGAUCAACAAUAAAGUUCAUGUAUGGCAA> hsa-mir-5591 MI0019151UGGGAGCUAAGCUAUGGGUAUACUGAGCUUAUGUAUGCAUCUGCAUACCCAUAGCUUAGCUCCCA> hsa-mir-5680 MI0019280
GCAUUGGGUUAGCAGGUUAGCCCAGCAUUUCCCUUCCUGGACACACAGGAGGAGAAAUGCUGGACUAAUCUGCUAAUCCAAUGC> hsa-mir-5681a MI0019281AGUUUUUGAAGAGUAUUGCCACCCUUUCUAGUCCCUAUUAGACUAGAAAGGGUGGCAAUACCUCUUCCAAAAACU> hsa-mir-5681b MI0019293GAAGAGGUAUUGCCACCCUUUCUAGUCUAAUAGGGACUAGAAAGGGUGGCAAUACUCUUC> hsa-mir-5682 MI0019282GGCCCAUGGGUCUUAUCCUGCAAGGUGCUGCAGAGACGAGGCCUGUAGCACCUUGCAGGAUAAGGUCUACUGGGCC> hsa-mir-5683 MI0019284GGAGCUUGUUACAGAUGCAGAUUCUCUGACUUCUUACUGCACCAGUGAAGUCAGGAUCUGCAUUUGAAUAAGACCC> hsa-mir-5684 MI0019285GCUGAACUCUAGCCUGAGCAACAGAGUGAGAUGGUCUUGUUUUGUUGCCCAGGCUGGAGUCCAGU> hsa-mir-5685 MI0019287CUCUACAUCACAGCCCAGCAGUUAUCACGGGCCCCUCCCCUCAAUGGGCCCGUGAUAACUGCAGGGCUGUGAUGUAGAG> hsa-mir-5686 MI0019290UAUCGUAUCGUAUCGUAUCGUAUUGUAUUGUACUGUAUUGUAUUGUACUGUAUUGUAUCGUAUCGUAUCGUAUCGUAUCGUA > hsa-mir-5687 MI0019291CCUCACUUAUCUGACUCUGAAAUCUUCUAAAUGGUACCCACUUUAUUUAGAACGUUUUAGGGUCAAAUAAGUACAGG> hsa-mir-5688 MI0019292GAAACACUUUGCCUUUUUACAGGAGUUUAUUAUGUUUGGA AUACACCUGUAGUCCUAGAUACUCAGGAGGGUGAGUAUCUAGGACUACAGGUGUGUGCUACCACGCU> hsa-mir-5690 MI0019295CUUUUAAUUUCAGCUACUACCUCUAUUAGGAUUUGGGAGUUAUACUAAUAGAGGUAAUAGUUGAAAUUAAGAG> hsa-mir-5691 MI0019296GGACAAGCUUGCUCUGAGCUCCGAGAAAGCUGACAGACAGCUGCUUGGUGUUCAGAGCUUGUCUGUCC> hsa-mir-5692a-1 MI0019297GACAGUACAAAUAAUACCACAGUGGGUGUACCUCAUGUGUGUACACCCUGUGAUAUUAUUUGUAAUAUC> hsa-mir-5692a-2 MI0019298UACAAAUAAUACCACAGUGGGUGUACCUCAUGUGUGUACACCCUGUGAUAUUAUUUGUA> hsa-mir-5692b MI0019311GAUAUUAUGAAUAAUAUCACAGUAGGUGUUCACACAUAAUGUGUACACCAUGUGUGUACACCCAUGUGAUAUUUGAAGUAGUAUGUC> hsa-mir-5692c-1 MI0019288UAUAACAUUGUAUAUACCCACUGUGAUAUUAAGAGUAAUAGCUCUCUAGGUUAUUAUGAAUAAUAUCACAGUAGGUGUACACAAUGUUGUA> hsa-mir-5692c-2 MI0019289UGUGUACACCAACUGUGAUAUUAGGAGUCCUAUUUAUUUUUAGGAUAUUAGGAAUAAUAUCACAGUAGGUGUACACA> hsa-mir-5693 MI0019300CUGGGAAGUUAGUUCAUUUCAGUCUGUGCUGUGAGCUAGCCAGCAGUGGCUCUGAAAUGAACUCAAACUCUAG> hsa-mir-5694 MI0019301GCCAACUGCAGAUCAUGGGACUGUCUCAGCCCCAUAUGUAUCUGAAGGCUGAGAAGUCCCAUGAUCCGCACUUGGC> hsa-mir-5695 MI0019302CAAGGCCUAUCUAUCUA GAUUCUUCUUGGCCUCUCUGAGCAUGCAUUCCUGAGACUCCAAGAAGAAUCUAGACAGAUAGGCCUUG> hsa-mir-5696 MI0019303GUGCUCAUUUAAGUAGUCUGAUGCCUACUACUGAUGACAUACAAUGUAAGUGCUCAUUUAGGCGUCAGACUACCUAAAUGAGCAC> hsa-mir-5697 MI0019304AGCAUAUUCUCAAGUAGUUUCAUGAUAAAGGGUGUAUGAGAGAUCAACCCUUUAUCAUGAAACGCUUGAGGAUACGCU> hsa-mir-5698 MI0019305CUGUGCACCUGGGGGAGUGCAGUGAUUGUGGAAUGCAAAGUCCCACAAUCACUGUACUCCCCAGGUGCACAG> hsa-mir-5699 MI0019306CUGUACCCCUGCCCCAACAAGGAAGGACAAGAGGUGUGAGCCACACACACGCCUGGCCUCCUGUCUUUCCUUGUUGGAGCAGGGAUGUAG> hsa-mir-5700 MI0019307UUAAUUAAUGCAUUAAAUUAUUGAAGGCCCUUGGGCACCCCAGGCCUUCAAUAAUUUAAUGCAUUUAUUGA> hsa-mir-5701-1 MI0019308GAUUGGACUUUAUUGUCACGUUCUGAUUGGUUAGCCUAAGACUUGUUCUGAUCCAAUCAGAACAUGAAAAUAACGUCCAAUC> hsa-mir- 5701-2 MI0019593GAUUGGACUUUAUUGUCACGUUCUGAUUGGUUAGCCUAAGACUUGUUCUGAUCCAAUCAGAACAUGAAAAUAACGUCCAAUC> hsa-mir-5702 MI0019309GCCUCAACUCCUGGGAUAUGUUGCUGAUCCAACCUGAAAUCCUUCUGUAGGUUGAGUCAGCAACAUAUCCCAUGACUUUUGGGU> hsa-mir-5703 MI0019310UUGCCGUCCCCUUCCUCGUCUUUUCCCCUCAGGAGAAGUCGGGAAGGUGGCGGCGG> hsa-mir-5704 MI00193 12UGAUCUUGUUUAGGCCAUCAUCCCAUUAUGCUAAGUCCAUGGGCAAACAUAACAGGAUGAUGGCCUAAACAAGACCA> hsa-mir-5705 MI0019313UCCCCAUUUACACAGGCCAUGAGCCCCGAAACACCCAUCCCAGGAUUGCUGAUGGGUGUUUCGGGGCUCAUGGCCUGUGUAAAUGGGGA> hsa-mir-5706 MI0019314AGCUAGGUCUUCUGGAUAACAUGCUGAAGCUUCUACGUCAUUCAGCACUUGCUUCAGCAUGUUUUCCAGAGGAUCUAGCU> hsa-mir-5707 MI0019315UGUAAGAACACGUUUGAAUGCUGUACAAGGCACAUAUGUGAACAUUGUACCACAUGUACAGCUUUCAAACAUGCUCUUAUA> hsa-mir-5708 MI0019316AUUACAGACAUGAGCGACUGUGCCUGACCAAAAGUCAACAUUAAACAACAAAUCUUGGCCAGGCACAGUGGCUCAUGCCUGUAAU

Claims (12)

Independently connecting the two nucleosides, formula (I) :.

A chirally controlled oligonucleotide containing one or more modified internucleotide bonds having the structure of
During the ceremony:
P ^* is an asymmetric phosphorus atom, Rp or Sp;
W is O, S or Se;
Each of X, Y and Z is independently -O-, -S-, -N (-L-R ¹ )-, or L;
L is a linear or branched chain C ₁ to C ₅₀ alkylene that may be covalently bonded or substituted, and one or more methylene units of L may be optionally and independently substituted C ₁ ~ C ₆ alkylene, C ₁ ~ C ₆ alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R') )-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R) ')-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-,-S (O) _2- , -S (O) ₂ N (R')-, -N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC ( Replaced by O)-or -C (O) O-;
R ¹ is a halogen, R, or optionally substituted C ₁ to C ₁₀ aliphatic, and one or more methylene units may be optionally substituted C ₁ to C. ₆ alkylene, C ₁ to C ₆ alkaneylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -SS-, -N (R')- , -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R') -, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-,-S (O) ) _2- , -S (O) ₂ N (R')-, -N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O) -Or-replaced by C (O) O-;
Each R'is independently -R, -C (O) R, -CO ₂ R, or -SO ₂ R, or:
Two R'on the same nitrogen together with their intervening atoms form a potentially substituted heterocyclic or heteroaryl ring, or two R'on the same carbon are them. Together with the intervening atoms of, it forms an aryl, carbocycle, heterocycle, or heteroaryl ring that may be substituted;
-Cy- is a optionally substituted divalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene;
_Each R is an independently optionally substituted group selected from hydrogen, or _C1-6 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl; and each.

Independently represents the binding to the nucleoside,
At least one of the modified internucleotide bonds is not a phosphodiester bond,
The oligonucleotide further comprises at least one or more phosphodiester bonds.
The oligonucleotide is an oligonucleotide having at least two consecutive modified nucleotide linkages .
The oligonucleotide is at least 15 nucleotides in length.
Oligonucleotide . The oligonucleotide according to claim 1, wherein the oligonucleotide is used.
The oligonucleotide is an oligonucleotide that independently comprises one or more modified internucleotide bonds having a structure of formula (I) in which -L- ^R1 is not a hydrogen atom. The oligonucleotide according to claim 1, wherein the oligonucleotide is used.
The oligonucleotide independently has the formula (Ib) :.

Containing one or more modified internucleotide bonds having the structure of
During the ceremony:
P ^* is an asymmetric phosphorus atom, Rp or Sp;
X is -O-, -S-, -N (-L-R ¹ )-, or L;
L is a linear or branched chain C ₁ to C ₅₀ alkylene that may be covalently bonded or substituted, and one or more methylene units of L may be optionally and independently substituted C ₁ ~ C ₆ alkylene, C ₁ ~ C ₆ alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R') )-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R) ')-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-,-S (O) _2- , -S (O) ₂ N (R')-, -N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC ( Replaced by O)-or -C (O) O-;
R ¹ is a halogen, R, or optionally substituted C ₁ to C ₁₀ aliphatic, and one or more methylene units may be optionally substituted C ₁ to C. ₆ alkylene, C ₁ to C ₆ alkaneylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -SS-, -N (R')- , -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R') -, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-,-S (O) ) _2- , -S (O) ₂ N (R')-, -N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O) -Or-replaced by C (O) O-;
Each R'is independently -R, -C (O) R, -CO ₂ R, or -SO ₂ R, or:
Two R'on the same nitrogen together with their intervening atoms form a potentially substituted heterocyclic or heteroaryl ring, or two R'on the same carbon are them. Together with the intervening atoms of, it forms an aryl, carbocycle, heterocycle, or heteroaryl ring that may be substituted;
-Cy- is a optionally substituted divalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene;
_Each R is an independently optionally substituted group selected from hydrogen, or _C1-6 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl;
each

Independently represents the binding to the nucleoside,
-Aligonucleotide in which L-R ¹ is not a hydrogen atom.
The oligonucleotide according to any one of claims 1 to 3.
Independently, equation (Ic) :.

Containing one or more modified internucleotide bonds having the structure of
During the ceremony:
P ^* is an asymmetric phosphorus atom, Rp or Sp;
L is a linear or branched chain C ₁ to C ₁₀ alkylene that may be covalently bonded or substituted, and one or more methylene units of L may be optionally and independently substituted C ₁ ~ C ₆ alkylene, C ₁ ~ C ₆ alkenylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -S-S-, -N (R') )-, -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R) ')-, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-,-S (O) _2- , -S (O) ₂ N (R')-, -N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC ( Replaced by O)-or -C (O) O-;
R ¹ is a halogen, R, or optionally substituted C ₁ to C ₁₀ aliphatic, and one or more methylene units may be optionally substituted C ₁ to C. ₆ alkylene, C ₁ to C ₆ alkaneylene, -C≡C-, -C (R') _2- , -Cy-, -O-, -S-, -SS-, -N (R')- , -C (O)-, -C (S)-, -C (NR')-, -C (O) N (R')-, -N (R') C (O) N (R') -, -N (R') C (O)-, -N (R') C (O) O-, -OC (O) N (R')-, -S (O)-,-S (O) ) _2- , -S (O) ₂ N (R')-, -N (R') S (O) _2- , -SC (O)-, -C (O) S-, -OC (O) -Or-replaced by C (O) O-;
Each R'is independently -R, -C (O) R, -CO ₂ R, or -SO ₂ R, or:
Two R'on the same nitrogen together with their intervening atoms form a potentially substituted heterocyclic or heteroaryl ring, or two R'on the same carbon are them. Together with the intervening atoms of, it forms an aryl, carbocycle, heterocycle, or heteroaryl ring that may be substituted;
-Cy- is a optionally substituted divalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene;
_Each R is an independently optionally substituted group selected from hydrogen, or _C1-6 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl; and each.

Independently represents the binding to the nucleoside,
When L is a covalent bond, R ¹ is not -H, an oligonucleotide. The oligonucleotide according to any one of claims 1 to 4 .
The oligonucleotide has a ribose sugar in which the 2'-OH group is replaced with a -OMe group.
Oligonucleotide. The oligonucleotide according to any one of claims 1 to 4 .
The above-mentioned oligonucleotide has a ribose sugar in which a 2'-OH group is replaced with a -OCH ₂ CH ₂ OME group.
Oligonucleotide. The oligonucleotide according to any one of claims 1 to 4 .
The oligonucleotide has a locked nucleic acid sugar,
Oligonucleotide. The oligonucleotide according to any one of claims 1 to 7 .
The oligonucleotide has a purity greater than 50%.
Oligonucleotide. The oligonucleotide according to any one of claims 1 to 8 .
The oligonucleotide has a purity greater than 80%.
Oligonucleotide. The oligonucleotide according to any one of claims 1 to 9 .
The oligonucleotide has a purity greater than 90%.
Oligonucleotide. The oligonucleotide according to any one of claims 1 to 10 .
The oligonucleotide is not bound to a solid carrier,
Oligonucleotide. A chirally controlled oligonucleotide composition comprising the oligonucleotide according to any one of claims 1 to 11 .

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